Hepatic encephalopathy (HE) is a neuropsychiatric syndrome in which cerebral function is impaired as a consequence of a previous liver failure. HE is present mainly in patients with liver cirrhosis. The symptoms of HE progresses through a wide range of cognitive and motor abnormalities, from mild cognitive impairment and motor incoordination to disorientation, sleep alterations, changes in personality, between other alterations. HE may lead to coma and death. Around 40%–60% of patients with liver cirrhosis present minimal HE (MHE), with psychomotor slowing, attention deficits, mild cognitive impairment and motor incoordination, which strongly reduce their ability to perform tasks of the daily life and increase the risk of accidents, falls and hospitalizations, reducing the life quality and span of the patients and posing an important economic burden to the health systems (Leevy et al., 2007; Bajaj et al., 2012; Stepanova et al., 2012; Felipo, 2013; Flamm et al., 2018; Häussinger, 2022). MHE affects several million people around the World and is a serious medical, social and economic problem. MHE is not evident, but may be detected using psychometric tests. MHE may progress to clinical HE, with evident symptoms, which may lead to coma and death (Felipo, 2013; Häussinger, 2022).

Currently there are no specific treatments for MHE. Only the use of rifaximin, a non-permeable antibiotic, has been approved to prevent the appearance of episodes of clinical HE (Bass et al., 2010). It is therefore necessary to develop new treatments to reverse the cognitive and motor alterations in patients with MHE. To design and test new treatments for MHE it is necessary to understand the molecular mechanisms by which liver injury leads to cognitive and motor impairment. Hyperammonemia and peripheral inflammation act synergistically to induce MHE (Shawcross et al., 2004; Felipo et al., 2012; Balzano et al., 2020a). The process leading to neurological impairment in MHE is being characterized and the underlying mechanisms are being identified in animal models of hyperammonemia and MHE. The general process by which liver injury induces MHE involves sequential induction of hyperammonemia and inflammation, which trigger neuroinflammation, leading to altered neurotransmission which finally impairs cognitive and motor function (see Cabrera-Pastor et al., 2019; Haüssinger et al., 2022; Balzano et al., 2023 for review and references therein). On the bases of the advances in the knowledge of the mechanisms by which liver injury induces MHE, several new treatments have been successfully tested to restore cognitive and motor function in animal models (Hernandez-Rabaza et al., 2015; Dadsetan et al., 2016a; Dadsetan et al., 2016b; Hernandez-Rabaza et al., 2016; Balzano et al., 2020b; Balzano et al., 2023).

The final inductor of the neurological impairment is the alteration of neurotransmission, mainly of GABAergic and glutamatergic neurotransmission (Cauli et al., 2006; Cauli et al., 2009a; Llansola et al., 2015). It has been proposed that altered GABAergic neurotransmission in cerebellum plays a relevant role in the pathogenesis of autism (Blatt, 2005), Huntington’s disease (Garret et al., 2018), Alzheimer’s disease (Luchetti et al., 2011; Assefa et al., 2018; Calvo-Flores Guzmán et al., 2018), Parkinson’s disease and multiple sclerosis (Luchetti et al., 2011) and in hyperammonemia and hepatic encephalopathy (Cauli et al., 2009b; Hassan et al., 2019; Arenas et al., 2022a; Arenas et al., 2022b).

The cerebellum is affected at early stages of HE, with altered cerebral blood flow, which correlates with impairment of performance in number connection test, bimanual coordination, visuo-motor coordination and in the Stroop test (Felipo et al., 2014). These functions are modulated by the cerebellum (Miall et al., 2001; Debaere et al., 2004; Pollock et al., 2007; Nabeyama et al., 2008; Segarra et al., 2008; Braga-Neto et al., 2012). Butz et al. (2010), also showed that ataxia, tremor and slowing of finger movements, functions also modulated by the cerebellum, are early markers of cerebral dysfunction at least in a subgroup of cirrhotic patients. Altered cerebellar function is therefore a main contributor to many early neurological alterations in cirrhotic patients.

Motor coordination is mainly modulated in cerebellum by circuits modulating synaptic integration involving both GABA inhibition and NMDA receptor activation (Watanabe et al., 1998). Hanchar et al. (2005) showed that small increases in a tonic GABA conductance in cerebellar granule neurons account for the adverse effects of ethanol on motor coordination, supporting that enhanced GABAergic activation in cerebellum impairs motor coordination. The association between GABAergic neurotransmission and motor incoordination induced by ethanol is further supported by the report of Hood and Buck (2000) showing that there is an association between allelic variations in the GABA _A receptor γ2 subunit and the genetic susceptibility to ethanol-induced motor incoordination. Also, mutation of the inhibitory ethanol site in GABA_A ρ1 receptors promotes tolerance to ethanol-induced motor incoordination (Blednov et al., 2017).

Woo et al. (2018) showed that augmentation of tonic GABA release by astrocyte-specific overexpression of MAOB resulted in motor incoordination, with a reduced motor performance in the rotarod. They propose that astrocytic tonic GABA release can control motor coordination by tonically inhibiting cerebellar neuronal excitability and that there is a dynamic control of motor coordination via selective modulation of the release, synthesis, and clearance of astrocytic GABA. Chiu et al. (2005) showed that mice lacking the GABA transporter subtype 1 (GAT1) show increased levels of extracellular GABA and reduced motor coordination as assessed in the rotarod as well as tremor and ataxia. In the same line, the GAT1 inhibitor tiagabine, a clinically useful antiepileptic drug, also induces motor incoordination, tremor and ataxia (Salat et al., 2015; Bresnahan et al., 2019).

The above studies and other reports in the literature show that motor coordination is mainly modulated by GABAergic neurotransmission in cerebellum.

There are sex-dependent differences both in motor coordination and in GABAergic neurotransmission under physiological conditions and also in the alterations induced by different pathological conditions in motor coordination and GABAergic neurotransmission.

These sex-dependent differences have been observed both in humans and in animal models.

In humans, young males have better bimanual coordination compared to their female counterparts (Shetty et al., 2014). Boys aged 6–9 years had higher scores than girls on eye-hand coordination (Canli et al., 2023).

There are also sex-dependent differences in the main proteins modulating GABAergic neurotransmission, including GABA_A receptors. Women have a higher GABA_A-benzodiazepine receptors availability than men across all brain regions: frontal, parietal, anterior cingulate, temporal and occipital cortices, and cerebellum (Esterlis et al., 2013).

Human males show higher levels of the α1 subunit of GABA_A receptors while older females showed lower α2, α5, and β3 subunit expression in superior temporal gyrus (Pandya et al., 2019). The authors propose that these changes might significantly influence GABAergic neurotransmission and lead to sex-specific disease susceptibility and progression.

Alcohol intoxication depresses neural activity by enhancing inhibition mediated by GABAergic neurotransmission. Brain then compensates with a compensatory reduction of GABA. Marinkovic et al. (2022) analysed the effects of binge drinking of alcohol on GABA levels in brain as analysed by magnetic resonance and found that GABA levels were higher in light drinkers women compared to light drinkers men.

Sex-dependent differences in motor coordination and GABAergic neurotransmission have been also reported in animal models. A sex-dependent deficit in motor coordination has been also reported in the HdhQ200/200 mouse model of Huntington’s disease. These mice show a reduction in motor coordination at 8 and 10 months of age, with a more severe phenotype in female mice (Cao et al., 2021). Similar worse effects in females than in males have been reported in human patients with Huntington’s disease (Hentosh et al., 2021).

In rats, female substantia nigra reticulata (SNR) neurons contained more KCC2 compared with age-matched males and this is associated with sex-dependent differences in GABA_A receptor function, promoting the sexual differentiation of the SNR (Galanopoulou et al., 2003). Kirson et al. (2021) reported that GABAergic spontaneous inhibitory postsynaptic currents (sIPSC) kinetics the central amygdala (CeA) differ between females and males. Moreover, ethanol increases sIPSC frequency in males but not in females.

GABAergic neurotransmission and motor coordination are also affected differentially in males and females by environmental contaminants. Low-level developmental lead exposure in mice induces male-specific reduction of motor coordination in the rotarod (Leasure et al., 2008). Cauli et al. (2013) also showed sex differential effects of developmental exposure to polychlorinated biphenyl 126 on motor coordination. PCB 126 impaired motor coordination at 2 months in males but not in females. Motor coordination was impaired by developmental exposure to endosulfan, cypermethrin, and chlorpyrifos in female rats but not in males. The effect of endosulfan and cypermethrin would be due to increased extracellular GABA in cerebellum, which remains unaltered in male rats (Gomez-Gimenez et al., 2018). Prenatal exposure to chlorpyrifos increases the expression of GABA_A receptor α1 subunit in females (Biosca-Brull et al., 2023).

A role for enhanced GABAergic neurotransmission in the pathogenesis of acute hepatic encephalopathy was proposed around 40 years ago by Schafer and Jones (1982). Schafer et al. (1984) observed that the pattern of the visual evoked potentials in rabbits with acute hepatic coma was identical to that in comas induced by drugs which activate GABAergic neurotransmission. They investigated the possible mechanisms by which GABAergic neurotransmission would be enhanced in acute hepatic encephalopathy and proposed that it would be due to increased levels of endogenous benzodiazepine ligands (Jones et al., 1993). They later proposed that hyperammonemia also contributes to enhance GABAergic neurotransmission by increasing the levels of neurosteroids and enhancing the binding of benzodiazepines to GABAA receptor and potentiating GABA-induced chloride currents (Jones and Basile, 1997; Jones, 2002).

We have later shown that alterations in GABAergic neurotransmission play a key role in the induction of motor and of some cognitive alterations in hyperammonemia and MHE (see below). Cauli et al. (2009b) showed, by brain microdialysis in freely moving rats that GABAergic neurotransmission is enhanced in cerebellum in vivo in rats with chronic hyperammonemia and MHE, but is reduced in cerebral cortex of the same rats. Similar findings were reported later in humans for cirrhotic patients with hepatic encephalopathy as analysed by transcranial magnetic stimulation (TMS). Hassan et al. (2019) using TMS showed an enhanced GABAergic neurotransmission in cerebellum of cirrhotic patients with HE in vivo. Groiss et al. (2019), also by TMS, showed a reduction of GABAergic neurotransmission of the motor cortex in vivo in patients with HE. This enhanced GABAergic neurotransmission plays a key role in the impairment of motor function and coordination, as discussed below.

The increase in GABAergic neurotransmission in cerebellum in hyperammonemia and MHE is a consequence of neuroinflammation. Hyperammonemia induces neuroinflammation in cerebellum, with activation of microglia and astrocytes, increased levels of TNFα and increased membrane expression of the TNFα receptor TNFR1 (Hernandez-Rabaza et al., 2016; Cabrera-Pastor et al., 2018; Balzano et al., 2020b). This results in enhanced activation of two signaling pathways which modulate GABAergic neurotransmission: the TNFα - TNFR1- NF-κB–glutaminase- GAT3 pathway and the TNFα - TNFR1- S1PR2 - CCL2 - BDNF - TrkB pathway.

The above results indicate that hyperammonemia-induced neuroinflammation enhaces the activation of the BDNF-TrkB pathway. This pathway, in turn is a main modulator of GABAergic neurotransmission under physiological conditions. Moreover, alterations in the BDNF-TrkB system contribute to altered GABAergic neurotransmission in different pathological situations. This suggested that the altered function of the BDNF-TrkB pathway would also alter GABAergic neurotransmission in hyperammonemia and MHE.

It has been shown that the BDNF-TrkB system regulates GABAergic neurotransmission by different mechanisms, modulating expression (Bulleit et al., 2000; Roberts et al., 2006), phosphorylation (Vithlani et al., 2013) and trafficking (Brünig et al., 2001; Tomoda et al., 2022) of GABA_A receptors, GABA release (Bardoni et al., 2007; Vaz et al., 2015), GABA transporter GAT-1 (Vaz et al., 2011), the function of the potassium-chloride co-transporter 2 (KCC2), which fine-tune intracellular chloride and the intensity of GABA_A receptor currents (Dai et al., 2014) and the expression of the GABA-synthesizing enzyme GAD67 (Arenas et al., 1996).

The net effect of BDNF-TrkB on GABAergic neurotransmission may be different for different cell types. For example, BDNF exposure decreased GABA responses in cultured mouse cerebellar granule cells through TrkB receptor-mediated signalling. However, the same system potentiates GABA responses in Purkinje neurons (Cheng and Yeh, 2005). The effects may be also different at different developmental stages. For example, the action of BDNF on GABA_A receptor currents changes from potentiating to suppressing during maturation of rat hippocampal CA1 pyramidal neurons (Mizoguchi et al., 2003).

Alterations in the BDNF-TrkB-GABA system has been proposed to contribute to different pathologies such as depression (Guilloux et al., 2012), neuropathic pain (Lever et al., 2003), stress-induced hyperalgesia (Dai et al., 2014), insomnia (Feng et al., 2019), panic disorder (Casarotto et al., 2010), epilepsy (Roberts et al., 2006), and also modulates neurogenesis (Vithlani et al., 2013), morphine reward (Koo et al., 2014) and sensory neurons (Pezet et al., 2002).

Enhanced activation of TrkB by BDNF affects several aspects of GABAergic neurotransmission in cerebellum of hyperammonemic rats (see Figure 2), including: increased levels of GAD65 and GAD67, the main enzymes synthesizing GABA; increased levels of total and extracellular GABA; increased membrane expression of GAT3 in activated astrocytes, which contributes to the increase of extracellular GABA, as discussed above; increased levels of gephyrin and of phosphorylation of the β3 subunit of GABA_A receptors, two of the main modulators of trafficking of these receptors, which result in increased membrane expression of GABA_A receptors. Hyperammonemia also alters the membrane expression of the K⁺-Cl^- cotransporters KCC2 and NKCC1, that are key modulators of the transmembrane chloride gradient and, therefore, of the intensity of the responses to activation of GABA_A receptors (Arenas et al., 2022b).

All these effects are reversed ex vivo in cerebellar slices from hyperammonemic rats by blocking TrkB with ANA12, indicating that all the above effects of hyperammonemia on GABAergic neurotransmission are mediated by enhanced function of the BDNF-TrkB system (Arenas et al., 2022b). Many of the above changes occur mainly in Purkinje neurons, leading to enhanced GABAergic neurotransmission, in agreement with the enhanced GABAergic tone in Purkinje neurons reported by Hassan et al. (2019) in cirrhotic patients with hepatic encephalopathy.

Enhanced GABAergic neurotransmission in cerebellum is responsible for motor incoordination in rat models of chronic hyperammonemia and hepatic encephalopathy.

A correlation between extracellular GABA levels in cerebellum in vivo and motor incoordination has been reported in rats with chronic hyperammonemia (Gonzalez-Usano et al., 2014). Moreover, reducing extracellular GABA by administration of pregnenolone sulfate restored motor coordination in hyperammonemic rats (Gonzalez-Usano et al., 2014).

Extracellular GABA in cerebellum of hyperammonemic rats may be also reduced by reducing neuroinflammation. Hernandez-Rabaza et al. (2016) showed that treatment with sulforaphane (1-Isothiocyanato-4-(methylsulfinyl)-butane) reduces neuroinflammation in cerebellum, leading to normalization of extracellular GABA and of motor coordination. Similar effects were obtained by treating the hyperammonemic rats with sildenafil (Agusti et al., 2017), by increasing extracellular cGMP (Cabrera-Pastor et al., 2018), by blocking peripheral TNFα with infliximab (Dadsetan et al., 2016b) or by blocking the S1PR2 with JTE-013 (Arenas et al., 2022b).

We have proposed that there is an interplay between GABAergic neurotransmission and neuroinflammation, which regulate each other (Agusti et al., 2017) and contribute to modulation of motor coordination and cognitive function. This interplay implies that enhanced GABAergic neurotransmission in some pathological situations (i.e., hyperammonemia, hepatic encephalopathy, Parkinson’s disease) may be reduced by reducing neuroinflammation, as mentioned above. This interplay also implies that neuroinflammation may be reduced in similar pathological situations by reducing GABAergic neurotransmission. This has been demonstrated in rats with chronic hyperammonemia and hepatic encephalopathy treated with bicuculline, an antagonist of GABA_A receptors, or with golexanolone, a GABA_A receptor-modulating steroid antagonist that reduces GABAergic neurotransmission by reducing the potentiation of GABA_A receptor activation by neurosteroids such as allopregnanolone (Johansson et al., 2015).