Juvenile mossy fibres: two pipers in the same pub?

According to an old Hungarian proverb, pub owners should not hire two pipers to play music simultaneously in their establishment. The potential cacophony could disturb the customers and decrease the pub's bottom line. This folk wisdom implies that in a single spatially restricted unit the existence of two different forces fulfilling the same general functional role could lead to unpredictable outcomes, which is not ideal for the proper function of the individual module. This general rule seems to hold true for a large portion of synapses – synaptic contacts using fast neurotransmitters are usually committed to either glutamatergic or GABAergic transmission (but see few notable exceptions: Hnasko & Edwards, 2012). In the current issue of The Journal of Physiology, Beltrán & Gutiérrez (2012) use a novel and innovative approach to demonstrate that developing hippocampal mossy fibres co-release glutamate and GABA, while at later developmental stages they use exclusively glutamate. The presence of machinery for the synthesis, storage and detection of GABA at juvenile mossy fibre terminals was demonstrated by the visualization of GABA, glutamic acid decarboxylase (GAD65/67), the vesicular GABA transporter (VGAT) and GABAA receptors by several laboratories in the last two decades (reviewed by Safiulina et al. 2010). Following these studies, the existence of direct GABAergic signalling between granule cells and CA3 pyramidal cells was demonstrated functionally by the stimulation of physiologically and pharmacologically identified mossy fibre tracks (Walker et al. 2001; Gutiérrez et al. 2003). The functional properties of GABA-mediated mossy fibre events in in vitro slices prepared from juvenile animals had all the hallmarks of mossy fibre signalling: the events showed marked paired-pulse and frequency facilitation, and they were blocked by mGluR agonists, while they were sensitive to GABAA receptor antagonists. These structural and functional observations together strongly suggested that until ∼postnatal day 20 mossy fibres can co-release glutamate and GABA (Walker et al. 2001; Gutiérrez et al. 2003). However, this concept was challenged by the lack of GABAergic component in unitary mossy fibre responses registered by paired recording of connected dentate granule cells and CA3 pyramidal neurones in cultured slices (Mori et al. 2004). In these experiments organotypic slices were prepared from 6-day-old pups and incubated for an additional 20 days, therefore direct comparison with data gained from acute slices is very difficult, as the exact developmental stage of these mossy fibres is hard to determine. Further evidence against developmentally regulated GABA and glutamate co-release was presented by Uchigashima et al. (2007). They demonstrated that monosynaptic GABAergic events can only be evoked with higher intensity stimuli and these events show differential sensitivity to group II and group III mGluR agonists, while at mature mossy fibre events both drugs can block synaptic transmission. The authors suggested that monosynaptic GABAergic events represent inputs from local interneurons which are activated with minimal stimulation, rather than GABA release from mossy fibre terminals. Given the complex nature of the neuronal network of the dentate gyrus and CA3 area, the later possibility is plausible – interneurons sending axonal or dendritic projections to the dentate gyrus could be activated with the minimal stimulation protocol. However, there was no potential explanation proposed to why this subset of interneurons could only be activated in a specific developmental time window. At the core of these opposing points of view lie the authors’ different take on the degree of surrounding network involvement in evoked mossy fibre responses. In order to solve this problem, Beltrán and Gutiérrez proposed a reasonable, yet somewhat radical, solution: the removal of the network. The authors mechanically isolated CA3 pyramidal cells from 2-week- and 3–4-week-old rats. In this preparation they could find functionally intact presynaptic terminals still attached to pyramidal cells. They used zinc staining and intracellularly transported fluorescent dyes to identify mossy fibre terminals. With small pipettes placed in close proximity to the visually identified boutons, they could evoke synaptic responses from large mossy fibre and small GABAergic terminals. Using this experimental design, the authors show that the stimulation of individual mossy fibre terminals leads to synaptic responses that show a high degree of facilitation upon increased stimulation frequency and they can be blocked with the group II mGluR agonist, DCG-IV. These events can be purely glutamatergic, GABAergic or mixed, while synaptic responses evoked by the stimulation of inhibitory terminals were always exclusively GABAergic. GABAergic and mixed mossy fibre signals only could be recorded from preparations from 2-, but not from 3–4-week-old animals. Data presented in this manuscript strongly support previous observations suggesting that in the first 3 weeks GABA and glutamate are co-released from rodent mossy fibre terminals. In principle, one could still argue that in the case of GABAergic and mixed mossy fibre events, small GABAergic terminals located on proximal dendritic shafts, close to the thorny excrescences that are innervated by mossy fibre terminals, were also activated by extracellular stimulation. However, this potential contamination would not disappear with age and the isolated GABAergic events would not show frequency facilitation. Future studies will determine how these complex events can influence the developing network and what kind of pathological changes could be expected in the absence of developmentally regulated GABAergic signalling in hippocampal mossy fibres.

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