The hippocampus dentate gyrus (DG) subgranular zone (SGZ) represents one of the neurogenic niches where radial glia-like neural stem cells (NSCs) continuously generate new neurons throughout adulthood. This process, known as adult neurogenesis, can be outlined as the stepwise progression of NSCs into progenitor cells, neuroblast fate specification, neuronal differentiation in dentate granule cells (GCs), survival, and their synaptic integration into the existing circuitry to participate in the hippocampal function [1
Neural activity affects multiple stages of hippocampal adult neurogenesis, and it is well established that the γ-aminobutyric acid (GABA) neurotransmitter plays a major role in mediating activity-dependent regulation of the neuronal development process, considering the early expression of GABA receptors (GABARs) by NSCs [3
]. Indeed, GABA, probably released by dentate parvalbumin-expressing interneurons, regulates NSC quiescence [4
], neuronal fate specification [5
], and synaptic neuron integration in the hippocampal circuit [6
]. Its functions are first mediated by tonic activation of extrasynaptic ionotropic GABAA
receptors onto newborn cells, and by phasic GABA activity when differentiating neurons receive GABAergic synaptic inputs from inhibitory interneurons of the subgranular zone and hilus [7
Tonic and phasic GABA activity on maturating neurons generate membrane depolarizations due to an increased intracellular chloride concentration produced by high and low expression of Na+
co-transporters (NKCC1) and K+
co-transporters (KCC2), respectively; as the neuronal maturation proceeds, a downregulation of NKCC1 and upregulation of KCC2 cotransporters occurs, thus dropping internal chloride concentration and making GABAergic signals hyperpolarizing [8
]. The depolarizing activity of GABA onto adult-generated granule cells seems to be crucial for their maturation, probably due to calcium entry through low-voltage activated T-type Ca2+
channels (T-type VDCC) [6
] precociously expressed by newborn cells.
In this context, we previously identified, in adult rat dentate gyrus, newborn granule cells at different stages of maturation, which exhibited peculiar morphological and electrophysiological properties [2
] and synaptic input as well [9
]. In line with literature evidence [10
], our findings showed that at a very early stage of newborn neuron maturation, the stimulation of medial perforant pathway (MPP) or hilus did not evoke any detectable synaptic response, indicating the absence of functional synapses. At eight-day post-mitosis, developing neurons exhibited GABAergic synaptic input, eliciting small amplitude responses with slow kinetics, and T-type VDCC expression in the cell membrane (maturation stage reported as Class 2 subclass I in [2
]). However, these very immature neurons also showed depolarized membrane potentials, likely inducing T-type VDCC inactivation, thus suggesting that GABA activity could not be able to promote increased calcium entry through T-type VDCC, as instead described for more mature newborn granule cells [10
Additionally, we observed, during current-clamp recordings of newborn neurons at this very early maturation stage, the occurrence of resting membrane potential oscillations were not induced by synaptic activities, but likely spontaneously originated (unpublished data). Compelling evidence in literature points out that voltage-dependent calcium channels and calcium-activated potassium channels cooperate to generate robust membrane potential oscillations in several experimental models, thus regulating the intracellular calcium concentration [11
Taking into account the above findings, we hypothesize that the spontaneous membrane potential oscillations recorded in 8-day old newborn granule cells (identified as Class 2 subclass I in [2
]) may promote calcium entry and that GABAA
R opening contributes to intracellular calcium level regulation, decreasing input resistance and dampening membrane potential oscillations. To address this issue, in the present paper, patch-clamp and calcium imaging techniques are used to record very immature granule cells of adult rat dentate gyrus. Our findings highlight a novel molecular and electrophysiological mechanism through which GABA fine-tunes intracellular calcium homeostasis in rat adult-born granule neurons at a very early stage of maturation.
In the present work, spontaneous membrane potential oscillations observed in very immature neurons of adult rat dentate gyrus were related to intracellular calcium level alterations and their modulation by GABA activity was investigated. The main results we found were the following: (i) resting membrane potential oscillations were able to elicite calcium entry into the cell, thus increasing intracellular calcium concentration; (ii) BK channels and T-Type VDCCs were involved in generating membrane potential oscillations; (iii) GABAA
R activation was able to decrease intracellular calcium level, probably shunting membrane currents and dampening resting membrane potential oscillations. Altogether, these findings outline a novel mechanism through which GABA regulates intracellular calcium homeostasis in rat adult-born granule neurons at a very early stage of their maturation (Figure 8
Our findings seem not to be consistent with the well-documented hypothesis that GABAergic activity induces intracellular calcium elevation in immature neurons [5
]. Such discrepancy may conceivably be explained, taking into account that in our experimental model, the recorded developing granule neurons show a resting membrane potential, definitely more depolarized compared to T-type VDCC threshold activation, thus inducing calcium channel inactivation. Therefore, although GABA is depolarizing, moving resting membrane potential towards the chloride equilibrium potential, there are not the electrical conditions needed for T-type VDCC opening and the resulting calcium influx. Of course, we cannot rule out the possibility that GABAergic depolarizing activity may activate T-type VDCCs when it occurs in conjunction with a hyperpolarizing spontaneous event or in less immature neurons showing a resting membrane potential more hyperpolarized compared to T-type VDCC threshold, as we previously demonstrated (Class 2 subclass III described in [2
Concerns on technical caveats in patch-clamp recordings of resting membrane potentials may arise considering, in particular, the high IR of immature neurons that might introduce an error leading to underestimating RMP values. However, this error can be avoided by performing a high resistance seal onto the cell membrane (approximately 10-times higher than cell IR), as described in the literature [20
]. Therefore, we believe our results are reliable because of the high resistance seals we obtained onto the membrane of the recorded immature neurons, as recommended in the literature. In addition, the maturation stage of newborn neurons we analyzed in this work was earlier than that of immature neurons considered in the investigations performed by other authors [10
], as can be inferred comparing their functional features.
The passive membrane properties and the depolarized resting membrane potential of the examined immature neurons induce the activation of a mechanism that generates membrane potential oscillations and calcium influx by involving BK channels. The BK channel is characterized by an exceptionally large single-channel conductance, and it can be synergistically activated by membrane depolarization and elevation of intracellular Ca2+
concentration, resulting in membrane repolarization and voltage-dependent Ca2+
channel closing to reduce Ca2+
entering the cell. However, the BK opening probability may be increased by the presence in the channel structure of β4 subunits, being able to shift the voltage dependence to more negative potentials [23
], and by the proximity of a calcium source. The expression of BK-β4 subunits in the plasma membrane of dentate granule cells was demonstrated by Brenner and colleagues [25
]. This result, together with our present findings, indicating a clustering between the BK channel and T-type VDCC, allow us to assert that the conditions to increase the BK channel opening probability exist in the immature neurons. In addition, the low intracellular calcium buffer, described in newborn dentate granule cells by Stocca and colleagues [26
], represents a further favorable condition for BK opening.
It is known that BK channels in mature dentate granule cells form complexes with NMDA receptors [27
], and this structural and functional coupling might also occur in immature neurons. In line with this, we found a robust BK current after local NMDA or glutamate application onto the recorded immature neurons, but ambient glutamate levels were not able to open NMDARs in our experimental conditions. Nevertheless, we cannot rule out a role of these receptors on membrane potential oscillations and intracellular calcium regulation in the intact brain.
Concerning the meaning of the electrophysiological and molecular mechanism we highlighted, it has to be taken into account that the features of the immature neurons examined in this work indicate their low probability of sending information in the hippocampal circuit, especially because of a very high threshold and low amplitude of action potential (see Figure 1
]). Therefore, the new granule neurons at this early stage of maturation, even though contacted, may not play a significant role in the hippocampal function. Thus, phasic and tonic GABA activity related to hippocampal activation might affect immature neuron developmental process, such as dendritic arborization, neuronal migration [28
], as well as cell survival, likely by influencing intracellular calcium fluctuations.
Along this line of reasoning, we previously demonstrated in the adult rat dentate gyrus that hippocampal circuitry activation, related to physical activity and behavioral experiences, promotes anticipation of GABA synaptogenesis and T-type VDCC appearance onto 1-week-old newborn neurons [9
] and that the survival of newborn cells was enhanced [31
]. Therefore, considering the present findings, we may speculate that GABA input activation, related to hippocampal function, prevents newborn cell death by modulating intracellular Ca2+
transients. Indeed, based on the mechanism here described, GABAA
R activation stabilizes the membrane potential at the Cl-
equilibrium potential, that in immature neurons is depolarized compared to the RMP, thus quenching the membrane potential oscillations generated by the coordinated activity of BK channel and T-type VDCC complexes during which calcium influx occurs.
In conclusion, our findings provide a significant contribution to understanding how adult-generated new neurons in the early stage of their maturation can regulate intracellular calcium levels, and highlight an unconventional role of GABA activity in intracellular calcium homeostasis. The electrophysiological and molecular mechanism here defined could also occur in immature neurons generated during brain development, which show functional features very similar to those of maturating neurons we have considered in this work and that have been described by Pedroni and colleagues in developing dentate gyrus [32
Finally, future experiments will be required to verify the implication of the described mechanism in promoting newborn neurons survival.