Tonic benzodiazepine-sensitive GABAergic inhibition in cultured rodent cerebellar granule cells
Introduction
GABA is the major inhibitory neurotransmitter in the mammalian CNS (Macdonald and Olsen, 1994, Mody et al., 1994, Rabow et al., 1995, Sieghart, 1995). It is found concentrated in presynaptic terminals, where it is compartmentalised within the lumen of synaptic vesicles. Following its liberation from these structures into the synaptic cleft, GABA produces inhibitory postsynaptic responses via the activation of various classes of postsynaptic GABA receptors (Mody et al., 1994). In the granule cells of the cerebellum, GABAergic inhibition is mediated solely via activation of postsynaptic GABAA receptors (Bisti et al., 1971, Vicini et al., 1986, Kaneda et al., 1995, Mellor and Randall, 1998). Quanta of GABA released from Golgi cell terminals (Eccles et al., 1966, Llinas and Walton, 1990) activate these postsynaptic GABAA receptors, thereby producing rapidly rising, exponentially decaying, inhibitory postsynaptic currents (IPSCs) (Vicini et al., 1986, Kaneda et al., 1995, Tia et al., 1996, Martina et al., 1997, Mellor and Randall, 1998).
Recent studies in rodent brain slices have revealed that cerebellar granule cells exhibit an additional constitutive form of GABAergic inhibition (Kaneda et al., 1995, Brickley et al., 1996, Tia et al., 1996, Wall and Usowicz, 1997). This is mediated by a tonically active background conductance arising from the activity of bicuculline-sensitive GABAA receptors. This novel form of inhibition has been shown to be capable of producing significant changes in the input impedance and excitability of granule cells (Brickley et al., 1996).
Developmental analyses of granule cells in rat brain slices have revealed age-dependent changes in the balance between the phasic and tonic forms of GABAergic inhibition. At early post-natal stages (i.e. around P7) classical, rapidly rising, exponentially decaying IPSCs are the dominant form of inhibitory synaptic input to cerebellar granule cells (Brickley et al., 1996, Tia et al., 1996, Wall and Usowicz, 1997). As the animals age, however, the contribution of the tonic form of inhibition steadily increases. In tandem with this, a decrease in both the frequency and duration of spontaneous IPSCs (sIPSC) is seen (Wall and Usowicz, 1997). By post natal day 40 the sIPSC frequency is close to zero, whereas the standing GABA-activated current averages close to 20 pA (Wall and Usowicz, 1997).
In cerebellar slices from immature animals, the transmitter responsible for the constitutive activation of GABAA receptors is thought to arise from spillover of vesicularly released GABA from the immediate confines of activated synapses. In more mature animals, however, a form of non-vesicular release seems to play a more prominent role; this may be through either reversed GABA transport or some other, as yet ill-defined process (Wall and Usowicz, 1997). In addition to underlying tonic interneuronal communication, evidence also points to a role of transmitter spillover in the production of slowly rising, slowing decaying components of phasic postsynaptic currents in cerebellar granule cells (Rossi and Hamann, 1998). Furthermore, in hippocampus, spillover of GABA can produce GABAB receptor-mediated presynaptic inhibition at nearby synapses (Isaacson et al., 1993).
The prominence and developmental progression of standing GABAergic conductances in cerebellar granule cells has been attributed to two factors. The first is the progressive postnatal development of glomerular structures at the site of synaptic input to granule cells. The second is the presence of high affinity postsynaptic receptors arising from the expression of the α6 subunit of the GABAA receptor. Although the presence of a large synaptic glomerulus is likely to promote spillover mediated phenomena, it is noteworthy that phenomena attributable to GABA spillover have been observed in slices and dissociated cultures of hippocampus where there is little or no evidence for the presence of synaptic glomeruli (Isaacson et al., 1993, Valeyev et al., 1993).
The full in vivo significance of constitutively active background GABAergic currents is presently unclear. Modelling of cerebellar circuitry, however, has suggested that granule cells require some form of tonic inhibitory conductance in order to effectively filter their excitatory input from the mossy fibres (Gabbiani et al., 1994). In addition, spillover of the excitatory transmitter glutamate has been proposed as a candidate mechanism for the mediation of long-lasting synaptic plasticity (Kullmann et al., 1996).
In this laboratory, as opposed to acute brain slices, we have often utilised primary cultures of mouse and rat cerebellum to study the properties of inhibitory synaptic inputs to cerebellar granule neurones (Mellor and Randall, 1997, Mellor and Randall, 1998). This cell culture system lacks the full synaptic circuitry of the cerebellum (for example, there are no Purkinje cells or mossy fibres present); however, granule cells excite and receive robust GABAergic feedback inhibition from GAD-positive cerebellar interneurones (presumably Golgi or possibly basket cells). In this respect, therefore, the inhibitory circuitry of granule cells in culture is resemblent of that of the intact cerebellum (Llinas and Walton, 1990, Wisden et al., 1996).
Although somewhat more distant from the in vivo situation, cerebellar cultures provide certain experimental advantages over tissue slices. For instance, paired cell recordings can be more readily made and drugs and other agents can be rapidly applied and removed. In addition, studies of granule cell maturation in vitro have indicated that developmental changes paralleling those in vivo can be produced under appropriate conditions (reviewed in Burgoyne and Cambray-Deakin, 1988, Wisden et al., 1996). For example, in murine cerebellar cultures grown in 5 mM K+ (but not 25 mM K+), expression of the α6 subunit of the GABAA receptor follows a similar pattern to that seen in vivo (Gao and Fritschy, 1995, Mellor et al., 1998). In this study we have examined whether another marker of granule cell maturation (Brickley et al., 1996, Wall and Usowicz, 1997), increased tonic GABAergic inhibition, also develops as granule cells age in primary culture. We found that, in addition to conventional phasic inhibition, a tonic GABAergic current is also present. This current is much larger in granule cells that receive a substantial inhibitory input and in addition is shown to be modulated by benzodiazepines.
Section snippets
Cell culture
Cerebellar cultures were prepared from post-natal day 5 Sprague-Dawley rat pups. Animals were killed under schedule 1 of UK Home Office regulations. The vermis and both hemispheres of the cerebellum were then dissected free and dissociated with trypsin to produce granule cell-rich cultures, as previously described (Randall and Tsien, 1995). Cells were plated onto matrigel coated coverslips and maintained in standard culture conditions (37°C, 5% CO2) for up to 4 weeks. The culture medium was a
Results
After 1 week or more in culture a substantial majority of cultured rat cerebellar granule cells exhibit spontaneously occurring synaptic currents (Randall et al., 1993). These consist of both glutamatergic EPSCs and GABAergic IPSCs. The former occur at much lower frequency and can be eliminated by application of CNQX (which was continuously present extracellularly throughout this study), whereas the latter can be reversibly and completely antagonised by both competitive (i.e. bicuculline) and
Discussion
Our results clearly indicate that cultured rat cerebellar granule cells exhibit a similar form of tonic GABAergic inhibition to that seen in cerebellar slices (Kaneda et al., 1995, Brickley et al., 1996, Tia et al., 1996, Wall and Usowicz, 1997). Similar currents have also been previously reported in cultures of embryonic hippocampus (Valeyev et al., 1993). The presence and properties of these currents in cultured granule cells provides a number of novel insights into their likely source.
Unlike
Acknowledgements
Supported by Fundação Coordenacão de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES) and the Medical Research Council (UK).
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- 1
Present address: Department of Biochemistry, UFMG, Belo Horizonte, Brazil.
- 2
Present address: Department of Physiology, USCF, San Francisco, CA, USA.