View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Neuron, Vol. 33, 625–633, February 14, 2002, Copyright 2002 by Cell Press Tonic and Spillover Inhibition of Granule Cells Control Information Flow through Cerebellar Cortex Martine Hamann, David J. Rossi, the fraction of granule cells excited by mossy fiber input and David Attwell1 to the cerebellum and, thus, to increase cerebellar infor- Department of Physiology mation storage capacity (Marr, 1969; Tyrrell and Will- University College London shaw, 1992). However, the relative importance of tonic Gower Street versus mossy fiber driven inhibition and their effect on London, WC1E 6BT the input-output relationship of the cerebellar cortex United Kingdom have not been studied, largely because until recently, it was not possible to block inhibition of granule cells without also blocking inhibition of Purkinje cells. Summary Here, we show that both spillover and tonic inhibition of adult granule cells are mediated largely by receptors We show that information flow through the adult cere- that are blocked by furosemide and insensitive to diaze- bellar cortex, from the mossy fiber input to the Purkinje pam and neurosteroids. We quantify the relative impor- cell output, is controlled by furosemide-sensitive, di- tance of tonic inhibition and of the direct and spillover azepam- and neurosteroid-insensitive GABAA recep- components of synaptically evoked inhibition. Finally, tors on granule cells, which are activated both tonically we demonstrate that tonic and spillover inhibition of and by GABA spillover from synaptic release sites. granule cells play a major role in controlling information Tonic activation of these receptors contributes a flow through the cerebellar cortex. 3-fold larger mean inhibitory conductance than GABA released synaptically by high-frequency stimulation. Results Tonic and spillover inhibition reduce the fraction of granule cells activated by mossy fiber input, generat- Tonic Inhibition of Granule Cells ing an increase of coding sparseness, which is pre- In cerebellar slices from adult rats, applying the GABAA dicted to improve the information storage capacity of receptor blocker bicuculline to granule cells whole-cell the cerebellum. clamped at Ϫ60 mV with electrodes containing isotonic Ϫ Cl (ECl ϭ 0 mV) produced an outward current shift of Introduction 42 Ϯ 4 pA, reflecting the suppression of a tonic conduc- tance of 700 Ϯ 60 pS generated by GABAA receptors Transmitter spilling out of the synaptic cleft can generate (Figure 1A). Similar results were seen with the GABAA ␮ a current in distant neurons if their receptors have a blockers GABAzine (Figure 1G) and picrotoxin (100 M, sufficiently high affinity (EC Ϸ 1 ␮M) to respond to two cells; data not shown). The current was not reduced 50 Ϯ ϭ ␮ the low concentration of transmitter reaching them; for (0.6% 2.9%, p 0.85) by TTX (1 M, Figures 1A and example, NMDA receptors in the case of glutamate 1C; cf. Kaneda et al., 1995; Wall and Usowicz, 1997) or 2ϩ Ϯ ϭ (Isaacson, 1999) or GABA receptors containing the ␣ by removing external Ca (9.6% 3.8%, p 0.09, A 6 2ϩ subunit (Rossi and Hamann, 1998). High-affinity recep- replaced by Mg and 2 mM EGTA; Figures 1B and 1C). Thus, the tonic conductance is not activated by GABA tors may also generate a current even in the absence released by action potentials or Ca2ϩ influx. of synaptic transmitter release if the background trans- mitter concentration maintained by transporters is in the Tonic Inhibition Is Mediated by Furosemide- low micromolar range that activates the receptors. Tonic Sensitive, Diazepam- and Neurosteroid- activation by low background levels of transmitter may Insensitive GABA Receptors occur for hippocampal and cerebellar NMDA receptors A In adult granule cells, most GABA receptors contain ␣ (Sah et al., 1989; Rossi and Slater, 1993) and in adult A 1 or ␣ subunits together with ␤ or ␤ and ␥ or ␦ subunits cerebellar granule cells that show an action potential- 6 2 3 2 (Laurie et al., 1992a; Quirk et al., 1994; Wisden et al., independent tonic GABA receptor conductance (Kaneda A 1996; Nusser et al., 1998, 1999; Pirker et al., 2000). Furo- et al., 1995; Tia et al., 1996a; Brickley et al., 1996; Wall semide (100 ␮M) inhibits receptors containing ␣6 sub- and Usowicz, 1997). units (with an IC of 10–40 ␮M), but not ␣ -containing In cerebellar granule cells, the molecular identity, cel- 50 1 receptors lacking ␣6 subunits (IC50 Ͼ 3 mM; Korpi et al., lular location, and functional role of the GABAA receptors 1995; Sigel and Baur, 2000), irrespective of the presence mediating spillover and tonic inhibition are uncertain. of ␥2 or ␦ subunits (Korpi and Lu¨ ddens, 1997; Thomp- Pharmacology shows that the receptors mediating spill- son et al., 1999). Furosemide produced an outward cur- ␣ over inhibition contain 6 subunits (Rossi and Hamann, rent (Figures 1D and 1G) that was 58% Ϯ 4% of the 1998), but the receptors’ other subunits are unknown. current produced by bicuculline in the same cells. No ␣ Mice lacking 6 subunits lack the tonic conductance current was produced by furosemide in bicuculline (Fig- (Brickley et al., 2001), but this need not imply that tonic ures 1D and 1G). Furosemide did not generate a current ␣ inhibition is due to receptors containing 6 subunits be- by blocking KCl cotransporters (Payne, 1997) and alter- cause of side effects of the ␣ knockout (see Discussion). Ϫ 6 ing [Cl ]i since the KCl cotransport blocker bumetanide Inhibition of granule cells has been postulated to reduce did not affect the membrane current (Figures 1E and 1G) when applied at a dose (220 ␮M) producing the as 100 ␮M (%80ف) 1Correspondence: [email protected] same inhibition of KCl transporters Neuron 626 Figure 1. Tonic Inhibition of Adult Granule Cells Is by Furosemide-Sensitive, Diazepam- Insensitive GABAA Receptors (A) Response of a granule cell at Ϫ60 mV (ECl ϭ 0 mV, so GABAA currents are inward) to bicuculline (40 ␮M) and to bicuculline in the presence of TTX (1 ␮M). Dotted lines show zero current. (B) Response to bicuculline in normal solution and in zero Ca2ϩ solution. (C) Mean bicuculline-evoked currents in the presence and absence of TTX (four cells) and of Ca2ϩ (four cells). (D) Response to furosemide (100 ␮M) and to furosemide in the presence of bicuculline (40 ␮M). (E) Response to furosemide and to bumeta- nide (220 ␮M). (F) Response to diazepam (0.5 ␮M) and to bicuculline. (G) Mean current shifts at Ϫ60 mV evoked by bicuculline (bic; 40 ␮M, 45 cells), GABAzine (10 ␮M, n ϭ 9), furosemide (furo; 100 ␮M, n ϭ 18), furosemide in bicuculline (n ϭ 4), bumetanide (bum; 220 ␮M, n ϭ 12), and diaz- epam (dz; 0.5 ␮M, n ϭ 7). Traces are filtered at 2 Hz, so individual spontaneous IPSCs are not visible. furosemide (Payne, 1997). Furthermore, as shown be- (Zhu et al., 1996; Wohlfarth et al., 2002; see Discussion). low, furosemide did not affect the holding current of In adult granule cells, the neurosteroid THDOC (3␣,21- Golgi, Purkinje, stellate, or basket cells that do not ex- dihydroxy-5␣-pregnan-20-1, 0.1 ␮M) generated an in- press ␣6 subunits (Laurie et al., 1992a), nor did it generate ward current (11 Ϯ 3 pA) corresponding to a 34% Ϯ 5% a current in granule cells from 6-day-old rats (n ϭ 5, potentiation of the bicuculline-sensitive tonic current in data not shown), at which age there is little expression the same cell (Figures 2A and 2C), which was abolished of ␣6 subunits (Laurie et al., 1992b). Diazepam (0.5 ␮M), by bicuculline (40 ␮M, n ϭ 4) and reduced 43% Ϯ 10% which potentiates 2- to 3-fold the response of ␣1-con- by furosemide (100 ␮M, n ϭ 5; data not shown). By taining receptors, but not of ␣6-containing receptors contrast, in granule cells in 6-day-old rats that do not (Saxena and MacDonald, 1996; Sigel and Baur, 2000), yet express ␦ subunits (Laurie et al., 1992b) and show did not affect the tonic current in adult granule cells no tonic current (Brickley et al., 1996; Wall and Usowicz, (Figures 1F and 1G), although it did potentiate (nonspillo- 1997), THDOC potentiated the response to 5 ␮M exoge- ver) synaptic responses mediated by ␣1-containing re- nous GABA by 84% Ϯ 15% (Figures 2B and 2C). Simi- ceptors in these cells (Figure 2H). Thus, the tonic current larly, pregnenolone sulfate (1 ␮M) did not affect the tonic in mature granule cells is generated exclusively by furo- GABAA current of adult granule cells (Figures 2D and semide-sensitive, diazepam-insensitive GABAA recep- 2F) but did inhibit the response of 6-day-old granule tors; i.e., receptors containing ␣6 subunits (see Dis- cells to 5 ␮M GABA (Figures 2E and 2F). Thus, compared cussion). to GABA-evoked currents in young animals, the tonic In granule cells, ␣6 subunits coassemble with ␥2 and/or current in adult granule cells is insensitive to neuroste- ␦ subunits (Nusser et al., 1998; Quirk et al., 1994; Jech- roids. The likely receptor subunit composition conferring linger et al., 1998). Neurosteroids modulate GABAA re- this pharmacological profile (furosemide-sensitive, diaz- ceptors, but, in receptors containing ␣6 or ␣1 subunits, epam- and neurosteroid-insensitive) is assessed in the this modulation is altered by the presence of ␦ subunits Discussion section. Tonic and Spillover Inhibition 627 Figure 2. Inhibition of Granule Cells by Neu- rosteroid-Insensitive GABAA Receptors (A) Potentiation of the tonic current in an adult (P35) granule cell by THDOC (0.1 ␮M); re- sponse to bicuculline shows size of tonic cur- rent; ECl ϭ 0 mV. (B) Potentiation by THDOC of the response of a young (P6) granule cell to GABA (5 ␮M).