GABAergic signalling to adult-generated neurons

Forty years after the first reports of adult neurogenesis initiated prolonged debate (Nottebohm, 2002), it is now widely accepted that newborn neurons are continuously produced in at least two discrete regions of adult mammals, including humans. The focus of debate has now shifted from supporting its existence to understanding the function and significance of adult neurogenesis in brain processes. In the olfactory bulb, constant incorporation of neurons from the subventricular zone (SVZ) may contribute to adaptive mechanisms for odour representations (Lledo & Saghatelya, 2005). In the hippocampus, newborn neurons generated by subgranular zone (SGZ) progenitors appear to play a role in network activity and some hippocampal-dependent behaviours (Saxe et al. 2006, 2007). In brain injury models, progenitors from the SVZ and SGZ migrate into non-neurogenic brain regions to assist in recovery (Lichtenwalner & Parent, 2006). The role of adult-generated neurons in normal and pathological brain function is a rapidly growing field of study propelled by technical advances in identifying and manipulating progenitors and newborn neurons. Recently, the amino acid GABA has emerged as a key regulator that controls multiple phases of adult neurogenesis. In the SVZ, GABA serves as a feedback regulator of neural production and migration (Bordey, 2007). In the SGZ, GABAergic mechanisms regulate differentiation and the timing of synaptic integration (Ge et al. 2007a). Here, we will briefly review current understanding of GABAergic signalling in hippocampal adult neurogenesis.

Mechanisms of GABAergic signalling

γ-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the adult brain. Synaptic inhibition is achieved when GABA released from presynaptic terminals causes a transient activation of postsynaptic receptors. A growing literature demonstrates that low ambient levels of GABA in the extracellular space can also persistently activate extrasynaptic GABA receptors, resulting in a phenomenon termed tonic inhibition (Farrant & Nusser, 2005). The specific actions and mechanisms of GABAergic signalling depend on distinct properties of the three classes of GABA receptors, termed GABA_A, GABA_B and GABA_C. GABA_A and GABA_B receptors are ubiquitously expressed throughout the brain. GABA_C receptors are similar to GABA_A receptors in their ion permeability, but have a distinct pharmacology and limited distribution.

GABA_B receptors are metabotropic G-protein-coupled receptors that are located at both pre- and postsynaptic sites. Activation of presynaptic GABA_B receptors reduces neurotransmitter release via inhibition of voltage-gated Ca²⁺ and release machinery. Activation of postsynaptic GABA_B receptors by synaptically released GABA can generate a slow inhibitory potential due to the activation of inward-rectifying potassium channels. By virtue of their coupling to G-proteins, GABA_B receptors are also poised to influence neuronal activity by modulating numerous intracellular signalling pathways.

In contrast, GABA_A receptors are ligand-gated ion channels that mediate fast synaptic inhibition by direct membrane hyperpolarization and shunting. Synaptic inhibition is primarily generated by GABAergic synapses located on dendrites, soma and the axon initial segment. GABA_A receptors are predominantly chloride-permeable ion channels with a slight permeability to bicarbonate ions. Thus, the polarity of the GABA_A-mediated response is determined by the resting potential of the cell and the Cl⁻ reversal potential. In mature neurons, GABA_A IPSPs are hyperpolarizing when the chloride reversal potential is more negative than the membrane resting potential. However, this relationship can be reversed in some types of neurons. For example, early in neuronal development the delayed expression of the K⁺–Cl⁻ cotransporter KCC2 results in elevated intracellular chloride, rendering GABA depolarizing (Owens & Kriegstein, 2002). During neuronal maturation the increased expression of KCC2 correlates with the shift from GABAergic excitation to inhibition. Regardless of the resting potential of the cell and the Cl⁻ reversal potential, GABA_A receptor-mediated responses can provide shunting inhibition as the increase in postsynaptic membrane conductance counteracts membrane depolarizations generated by concurrent excitatory events.

Step-wise maturation of adult-generated granule cells

Adult neurogenesis is a multi-step process that encompasses the proliferation of progenitors, differentiation of newborn neurons and the maturation of newborn neurons that incorporate into the existing neural circuit. In the dentate gyrus, immunohistochemical and transgenic methods have provided extensive information about markers of differentiation and morphology of adult-generated dentate granule cells (DGCs) during their maturation. Retroviral labelling has provided detailed insight into the temporal progression of maturation. Work from many groups using these methods has demonstrated that nestin-expressing neural stem cells with a radial glial morphology asymmetrically divide to produce transit-amplifying cells. This heterogeneous population of progenitors begins to display some neuronal properties including tonic activation of GABA_A receptors and the first GABAergic synapses (Tozuka et al. 2005; Ge et al. 2006).

As newborn DGCs further differentiate, they send axonal projections that may reach CA3 within a week of cell division (Hastings & Gould, 1999). DGCs at this early postmitotic stage are selectively labelled in proopiomelanocortin (POMC)-EGFP transgenic mice. POMC protein is not expressed in dentate granule cells, but a fragment of the gene reliably directs the expression of EGFP to newborn DGCs (Overstreet et al. 2004). The transient expression of EGFP provides a ‘snapshot’ of newborn DGCs at an early developmental stage at any age of the mouse (Fig. 1). Newborn neurons at this stage are located in the inner granule cell layer and their axons project through the hilus to CA3. Their short dendrites reach through the granule cell layer but do not extend through the entire molecular layer and are devoid of spines. Birth dating with bromodeoxyuridine (BrdU) indicates that maximal POMC-GFP expression occurs at ∼12 days after BrdU incorporation, with a broad range (3–24 days) that suggests heterogeneity in the timing of maturation (Overstreet-Wadiche et al. 2006a). At this early developmental stage, adult-generated granule cells receive exclusively GABAergic synaptic input (Espósito et al. 2005; Overstreet Wadiche et al. 2005; Ge et al. 2006). GABAergic synaptic events have immature characteristics including slow rise and decay phases and depolarized reversal potentials (Fig. 2). Compared with neighbouring mature granule cells, synaptic currents in newborn granule cells are also relatively insensitive to the α1 subunit-selective GABA_A receptor modulator zolpidem, suggesting that lack of this developmentally regulated subunit may contribute to the slow kinetics of synaptic currents (Overstreet Wadiche et al. 2005; Karten et al. 2006). Slow GABA_A receptor-mediated activity is thought to originate from dendritic locations whereas fast synaptic responses arising from perisomatic GABAergic synapses do not appear until well after glutamatergic synapses have been established (Espósito et al. 2005). This sequence of synaptogenesis recapitulates the integration of DGCs during neonatal development and results in a similar pattern of innervation regardless of time of birth (Laplagne et al. 2007).

Although newborn granule cells labelled with POMC-EGFP express functional AMPA and NMDA receptors (Fig. 2; Overstreet Wadiche et al. 2005), dendritic spines and glutamatergic synaptic input have not been detected at this early stage. Consistent with these results, retroviral studies demonstrate that DGCs extend dendrites through the molecular layer to receive synaptic input from the perforant path during the second and third postmitotic weeks (Espósito et al. 2005; Ge et al. 2006; Zhao et al. 2006). Mounting evidence indicates that during this developmental period immature DGCs exhibit a striking propensity for synaptic plasticity, suggesting that they could make a unique contribution to experience-dependent modification of the mature neural circuit. Immature granule cell spine mobility is enhanced (Zhao et al. 2006) and accompanied by a low threshold for stimulation-induced long-term synaptic potentiation (LTP), a cellular correlate of learning (Schmidt-Hieber et al. 2004; Ge et al. 2007b). Ge et al. (2007b) also demonstrated that granule cells between 4 and 6 weeks old display an increased magnitude of LTP compared to both younger and older granule cells. It is during this period of excitatory synaptic integration and enhanced synaptic plasticity that fast GABA_A receptor synaptic input is established (Espósito et al. 2005). This may represent a transition in the function of GABAergic input from a developmental trophic signal to its mature role of controlling neuronal output.

Although the sequence of DGC integration is similar in the adult and in the developing hippocampus, the rate of maturation is delayed in adults (Overstreet-Wadiche et al. 2006a; Zhao et al. 2006). This delay may be related to a developmental-related alteration in network activity. The neonatal hippocampal network exhibits a characteristic pattern of spontaneous activity that declines after the second postnatal week and is thought to be important for appropriate hippocampal development (Ben-Ari, 2001). In support of the notion that this pattern of network activity promotes neuronal maturation, suppressing neonatal spontaneous network activity delays the maturation of POMC-EGFP-labelled DGCs in neonates (Overstreet-Wadiche et al. 2006a). Other conditions also modulate the rate of DGC maturation in adults, including ageing (Rao et al. 2006), antidepressant treatment (Wang et al. 2008) and seizures (Overstreet-Wadiche et al. 2006b). These results indicate that the timing of granule cell maturation, as well as progenitor proliferation, is subject to regulation.

GABAergic mechanisms regulating adult neurogenesis

Identification of GABAergic signalling to progenitors and newborn neurons in the adult dentate gyrus raises the possibility that hippocampal network activity is initially translated to newly generated cells via GABAergic depolarization. Despite its classical function of neuronal inhibition, it is well established that GABA acts as a trophic signal in the developing nervous system (Owens & Kriegstein, 2002). For example, during embryogenesis, the early formation of depolarizing GABA_A receptor-mediated activity triggers voltage-gated Ca²⁺ influx that in turn regulates DNA synthesis (LoTurco et al. 1995). Likewise, GABAergic depolarization of adult-generated progenitors promotes neural development by driving pro-neural gene expression including NeuroD (Tozuka et al. 2005). Depolarization by GABA thus provides a mechanistic basis for in vivo ‘excitation–neurogenesis coupling’, or the ability of hippocampal progenitors to respond to network activity by promoting neural differentiation (Deisseroth et al. 2004). GABAergic depolarization also promotes the functional integration of adult-generated DGCs. In an elegant study using retroviral techniques, Ge et al. (2006) showed that genetically switching GABAergic depolarization to hyperpolarization by interfering with the Cl⁻ importer NKCC1 in adult-generated DGCs reduced dendritic arborization and delayed GABAergic and glutamatergic synapse formation. Together these data suggest the possibility that GABAergic depolarization provides a general mechanism to promote the maturation and synaptic integration of newborn neurons in response to network activity.

Future directions

Mounting evidence supports a role for GABAergic signalling in the development and integration of adult-generated neurons. Future studies are needed to unravel the specific contribution of GABAergic mechanisms in the regulation of neural differentiation, survival and integration in response to physiological and pathological stimuli such as exercise, enrichment and seizures. A full understanding will require detailed knowledge of the properties of tonic and phasic GABAergic activity and the resulting intracellular signalling cascades that drive depolarization-induced regulation. Presumably such regulation occurs in a developmental stage-specific manner. As intracellular chloride concentrations distinguish adult from newborn DGCs, much work focuses on the chloride-permeable GABA_A receptor, whereas involvement of GABA_B receptors in adult neurogenesis has been largely unexplored. GABA_B receptor-mediated activation of inward-rectifying K⁺ channels is hyperpolarizing regardless of chloride gradients, predicting postsynaptic GABA_B receptors would counteract GABA_A-mediated depolarization. However, GABA_B receptor-mediated K⁺ responses are absent in newborn granule cells (our unpublished data). Whether newborn neurons lack functional GABA_B receptors, GIRK channels or coupling between the two is under investigation. Altogether, the slow phasic and tonic activation of GABA_A receptors on newborn granule cells, along with the lack of GABA_B receptor-mediated hyperpolarization suggest that GABAergic signalling is optimized for depolarization-mediated trophic functions.