The paper from Szabadics and colleagues contests two dogmas of GABA neurotransmission: 1) that in the adult brain, the effects of GABA synapses are inhibitory; and 2) that among GABAergic interneuron subclasses, axo-axonic cells (AACs) are probably the most powerfully inhibitory.
Like most GABAergic interneuron subclasses, AACs utilize GABA as their main neurotransmitter and the effects of GABA at AAC synapses are mediated by GABAA receptors, which after binding GABA open a chloride channel. Usually, the opening of GABAA receptor chloride channels produces hyperpolarization and thus inhibition. AACs are unique because they make synapses onto the initial segment of the pyramidal cell axon, close to the site where action potentials are initiated. Because action potential initiation requires depolarization, it was thought that AAC-mediated hyperpolarization was strategically placed: The closer an inhibitory synapse is to the site where action potentials are initiated, the more powerful the inhibition. The claim that AACs were more powerful than other interneurons to inhibit pyramidal cell firing was never verified experimentally. However, very few scientists would have doubted that AAC synapses have an inhibitory effect (Howard et al., 2005).
The surprising findings of Szabadics and colleagues show that after releasing GABA, AACs produce depolarization, instead of hyperpolarization of the pyramidal cell membrane. Moreover, the results show that AAC-mediated depolarization is so powerful that in more than 50 percent of the cases in which a single AAC is stimulated, it depolarizes the postsynaptic pyramidal neuron to the point of making it fire an action potential. This suggests that single AACs can produce much more excitation than can single pyramidal neurons, because single pyramidal neurons rarely, if ever, can make a postsynaptic neuron fire spikes.
The reason AACs are so powerfully excitatory is the same reason they were thought to be strongly inhibitory: They make synapses very close to the action potential initiation site. However, instead of hyperpolarization, the AAC synapses produce depolarization, because (as Szabadics and colleagues show) the chloride concentration gradient at AAC synapses determines a depolarizing, rather than hyperpolarizing effect of GABA.
Like AACs, synapses made by pyramidal cells (the prototypical excitatory neurons of the cerebral cortex) have a depolarizing effect. Unlike AACs, however, pyramidal cell synapses are electrically "far" from the site of action potential initiation. This happens because when depolarizing synaptic signals travel within neurons, they typically attenuate in size. Thus, the longer the distance they need to travel to reach the action potential initiation site, the weaker their electrical effect is. In the case of the AAC synapses, the distance is very short, and thus, the effect of these synapses (now shown to be excitatory) is strong.
Depolarizing effects of GABA have been previously found in very immature neurons, where they have been associated with maturational plasticity of GABA synapses (Kandler, 2004). However, until now in the mature brain, the chloride concentration gradient was thought to determine membrane hyperpolarization everywhere in the cell. Szabadics and colleagues provide data consistent with a potential mechanism that can explain the differential chloride gradient: the differential localization of the chloride transporter KCC2, which is almost absent close to the AAC synapses. However, whether or not this mechanism completely accounts for the differential chloride gradient is somewhat irrelevant, because the physiological data are strong on their own.
I think the reason these findings were not reported before by other groups is mainly because, from a number of different points of view, the experiments performed by Szabadics and colleagues are technically challenging. Discovery of the excitatory effect of AACs required first of all the study of small circuits of neurons in living brain slices in which at least one of the cells belonged to the AAC population and had intact connections with nearby pyramidal cells. This is already challenging, because AACs are not easy to identify prior to recording and are not the most abundant class of GABAergic interneuron. In addition to the low probability of finding AACs, in order to study cells without perturbation of the normal chloride gradients, the researchers had to use perforated patch clamp recording, which is considerably more difficult than standard recording techniques.
A very important aspect of this study is that, although the main findings were observed in cortical tissue obtained from the brain of young rats, the authors report essentially identical results from studying adult human cortical neurons. I think this is very important, because it shows that at least in this respect, the rat cortex is an excellent model for further studies of the role of AACs in cortical microcircuits.
GABA transmission mediated by AACs is thought to be altered in the brain of subjects with schizophrenia. The present data from Szabadics and colleagues are thus highly relevant for current models on the neurobiology of schizophrenia (Lewis et al., 2005). If the findings of Szabadics and colleagues are replicated by other groups, the models that propose a role for AACs in the biological basis of schizophrenia will have to be substantially modified. Being able to use the rat (or mouse) brain as a valid animal model to study this role is tremendously important.
Clearly, before jumping to quick conclusions about the therapeutic effects of pharmacological manipulations of AAC cell-mediated excitation, we need to learn more about the role of AACs in the normal brain. In particular, although individual AACs may have strong excitatory effects, it is not clear that AACs as a neuronal population could produce more excitation than the network of pyramidal cells. Indeed, in the cortex only about 20-30 percent of the neurons are GABAergic interneurons; the remainder are pyramidal cells. Moreover, AACs are a subpopulation within the subpopulation of parvalbumin-containing interneurons (parvalbumin is a calcium-binding protein whose expression by these neurons seems to be altered in schizophrenia). Thus, whereas each pyramidal cell receives synaptic input from hundreds, if not thousands, of other pyramidal cells, it receives input from only 2-4 AACs. This suggests that the main excitatory drive for pyramidal cells comes from other pyramidal neurons and that AAC-mediated excitation may have more selective and subtle roles. Consistent with this idea, recent experiments in living rats suggest that AACs do not discharge at the same time as pyramidal cells, and therefore that AACs are activated in a very precise and selective manner (Klausberger et al., 2003; Somogyi and Klausberger, 2005).
View all comments by Guillermo Gonzalez-Burgos