Abstract
Colombi I, Rastogi M, Parrini M, Alberti M, Potenzieri A, Chellali MM, Rosati S, Chiappalone M, Nanni M, Constabile A, Cancedda L. iScience 2024;27:109438. Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in adults. Depolarizing GABA responses have been well characterized at the neuronal-population average level during typical neurodevelopment and partially in brain disorders. However, no investigation has specifically assessed whether a mosaicism of cells with either depolarizing or hyperpolarizing/inhibitory GABAergic responses exists in animals in health/disease at diverse developmental stages, including adulthood. Here, we showed that such mosaicism is present in wild-type (WT) and Down syndrome (DS) neuronal networks, as assessed at increasing scales of complexity (cultures, brain slices, behaving mice). Nevertheless, WT mice presented a much lower percentage of cells with depolarizing GABA than DS mice. Restoring the mosaicism of hyperpolarizing and depolarizing GABA-responding neurons to WT levels rescued anxiety behavior in DS mice. Moreover, we found heterogeneous GABAergic responses in developed control and trisomic human-induced pluripotent stem-cell-derived neurons. Thus, a heterogeneous subpopulation of GABA-responding cells exists in physiological/pathological conditions in mouse and human neurons, possibly contributing to disease-associated behaviors.
Commentary
The concept that gamma-aminobutyric acid type A (GABAA) receptor-mediated synaptic transmission is depolarizing rather than hyperpolarizing early in life has become nearly a dictum in neuroscience. Pioneered by Ben-Ari and many others since the early 1990s, the idea is that there is an age window during which GABA is depolarizing, with this “paradoxical” action critical for normal developmental and trophic processes. 1 Subsequently, a solid body of data supports GABA-related depolarizing activity beyond the early developmental window in many pathological brain states, especially epilepsy and neurodevelopmental disorders. The reason for GABA-induced depolarization is an expression or function of the cation-chloride co-transporters that govern chloride (Cl−) homeostasis. NKCC1, a chloride importer, is preferentially expressed early in development and keeps the intracellular Cl− concentration high, so that when GABA binds to its type A receptor, Cl− effluxes and depolarizes the neuron, sometimes enough to contribute to neuronal excitation. On the other hand, KCC2 is a Cl− exporter, keeping intracellular Cl− concentration low, so that GABAA receptor activation causes Cl− influx and hyperpolarization. The ratio of NKCC1/KCC2 at a given developmental stage determines the polarity of Cl− movement upon GABAA receptor activation; the timing and extent of GABA responses switching from depolarizing to hyperpolarizing is a critical feature of neuronal maturation. However, in pathological states, and even in normal animals, depolarizing responses to GABA may persist in a finite subset of neurons or in certain cell regions. This potential flexibility in GABA response polarity may have significant functional or pathophysiological implications.
In trisomy 21 (Down syndrome [DS]), it has long been suspected that the balance between excitation and inhibition in the brain favors excessive inhibition, thought to explain some of the associated cognitive, learning, and memory deficits. 2 Evidence for this conclusion derives from pathological specimens that show an increased density of inhibitory interneurons and decreased density of excitatory primary neurons. However, up to 10% of individuals with DS have epilepsy, and the incidence of one form of childhood epilepsy, infantile epileptic spasms syndrome (IESS), is 100 times greater than the general population. 3 Therefore, an apparent paradox seems to be present—how could excessive inhibition in DS lead to the increased seizure predisposition, since seizures typically entail increased excitation or decreased inhibition? The authors of the current commentary set out to determine whether persistent depolarizing GABA responses are present in an animal model of DS, since GABA dysfunction could be involved in various clinical aspects of DS such as the high incidence of anxiety disorder. 4 While not explicitly exploring seizure mechanisms in this paper, the potential involvement of persistent GABA depolarization would be clearly relevant to seizure predisposition. The team already showed that in the Ts65Dn mouse model of DS, GABA depolarization persists well into adulthood, with correlated excessive persistence of NKCC1. 5 Furthermore, they demonstrated that pharmacological inhibition of NKCC1 with bumetanide or knockdown of NKCC1 could rescue some core cognitive deficits in DS models and in tissue from humans with DS.4,6
The mechanisms of epilepsy in DS syndrome are not fully understood. The seizure risk is increased for at least two broad reasons—the medical comorbidities that accompany DS (eg, heart disease, increased incidence of infection, immune dysregulation, and increased incidence of leukemia) and the structural and network abnormalities inherent in brain maldevelopment in DS (eg, dendritic spine dysgenesis, abnormal dendritic arborization, and anomalous cortical lamination). 7
The Ts65Dn model has been used extensively to study DS pathophysiology. Several neurological features of the Ts65Dn mouse mimic human DS, including impaired cognition and synaptic plasticity. While spontaneous seizures have not been documented in Ts65Dn mice (nor in any other DS animal model), in response to the gamma-aminobutyric acid type B (GABAB)-R agonist ɤ-butyrolactone (GBL), Ts65Dn mice develop epileptic spasms and electroencephalography features (polyspike-wave bursts and electrodecrements) of IESS plus response to IESS treatments including adrenocorticotrophic hormone and vigabatrin. 8 The mechanism of hyperexcitability is overexpression of the G protein-coupled potassium channel subunit GIRK2, suggesting that at least part of the seizure predisposition in DS could be enhanced GABAB inhibition.
Here, a complementary hypothesis is explored—persistent GABAA-mediated depolarization in Ts65Dn. 4 The investigators used cultured hippocampal neurons from wild-type (WT) or Ts65Dn animals to investigate GABA polarization as a function of age and disease. They found that both WT and Ts65Dn neurons responded to GABA stimuli by depolarizing, even after such GABAergic depolarizing responses should have abated. These depolarizing GABA responses were demonstrated at several levels of complexity, from neuronal cultures to brain slices to whole animals. A much higher percentage of Ts65Dn neurons had depolarizing GABA responses than WT neurons.
In the first set of experiments, cultured neurons were monitored using a calcium-sensitive dye to measure the depolarizing action of GABA. The addition of GABAA agonists led to depolarizing responses as represented by increased calcium signal. Over development in culture (which has an uncertain correlation with age in vivo), these responses persisted. The NKCC1 inhibitor, bumetanide, reduced the percentage of cells with depolarizing GABA responses. The authors concluded that some depolarizing GABA neurons persisted in both Ts65Dn neurons and euploid cells, but a lot more in Ts65Dn.
The investigators then monitored neuronal firing using multi-electrode arrays in both cultures and cortical slices from adult mice and again documented a significantly higher percentage of Ts65Dn neurons with GABA depolarizing responses. Furthermore, GABA depolarizing responses were monitored in hippocampal neurons in adult freely moving mice using calcium imaging. The GABAA receptor allosteric modulator diazepam reduced calcium events, consistent with the presence of neurons with depolarizing GABA responses; the NKCC1 inhibitor bumetanide further reduced calcium events. In behavioral tests of anxiety in adult Ts65Dn mice, diazepam exacerbated anxiety that could be rescued by bumetanide. Diazepam did not exacerbate seizures in either WT or Ts65Dn mice in this study or others, 9 suggesting that persistent GABA depolarization did not reach an overt pathophysiological level. Yet, detailed mechanisms remain to be elucidated.
Finally, for translational proof-of-principle, additional experiments using trisomy 21 induced pluripotent stem cells (iPSCs) derived from a patient with mosaic DS supported their hypothesis—14% of neurons in control cultures, and 62% of neurons from DS cultures displayed depolarizing GABA responses; the latter were rescued by bumetanide, supporting the contention that this NKCC1 blocker might have clinical utility.
The authors recognize several limitations of their study, including the definition of “maturity” of neurons in culture and how well time in culture correlates with actual age. They suggest that proof of persistent GABA-mediated depolarization would require long-term tracking of individual neurons from early development to full maturation. Regarding their use of cultures, brain regional differences could complicate some of the results, as opposed to pure cultures of the hippocampus, cortex, etc. Moreover, concerns about genetic background were raised, and recent reports question whether the Ts65Dn model is actually the best model of human DS. 10 Forty-five genes are present in Ts65Dn mice that are not present on human chromosome 21, raising the possibility that these additional genes in Ts65Dn mice are contributing to the neurological deficits described. Therefore, Ts65Dn mice may be less directly comparable to human DS. Newer models, such as one with a genetically engineered extra human chromosome 21 in mice (called transchromosomic artificial chromosome 21 [TcMAC21]), harbor a genetic repertoire more similar to human DS and also display GBL-induced epileptic spasms. 11
In conclusion, Colombi et al 4 show that there exists a subpopulation of neurons that respond to GABA by depolarizing, long after the expected switch to hyperpolarizing GABA responses, in neurons from WT animals, Ts65Dn mice, and human iPSCs. Across this scale of increasing complexity, the proportion of neurons with GABA-mediated depolarization is significantly higher in Ts65Dn mice than in euploid controls, attesting to increased neuronal excitability in this model of DS and possibly explaining their enhanced seizure susceptibility. These findings could also underlie enhanced seizure susceptibility in other neurodevelopmental and acquired disorders and might lead to novel therapeutic strategies.
