Depression of Glutamate and GABA Release by Presynaptic GABA

Presynaptic GABA B receptors (GABA B R) control glutamate and GABA release at many synapses in the nervous system. In the present study we used whole-cell patch-clamp recordings of spontaneous excitatory and inhibitory synaptic currents in the presence of TTX to monitor glutamate and GABA release from synapses in layer II and V of the rat entorhinal cortex (EC) in vitro. In both layers the release of both transmitters was reduced by application of GABA B R agonists. Quantitatively, the depression of GABA release in layer II and layer V, and of glutamate release in layer V was similar, but glutamate release in layer II was depressed to a greater extent. The data suggest that the same GABA B R may be present on both GABA and glutamate terminals in the EC, but that the heteroreceptor may show a greater level of expression in layer II. Studies with GABA B R antagonists suggested that neither the autonor the heteroreceptor was consistently tonically activated by ambient GABA in the presence of TTX. Studies in EC slices from rats made chronically epileptic using a pilocarpine model of temporal lobe epilepsy revealed a reduced effectiveness of both autoand heteroreceptor function in both layers. This could suggest that enhanced glutamate and GABA release in the EC may be associated with the development of the epileptic condition. Copyright © 2006 S. Karger AG, Basel Received: October 18, 2006 Accepted after revision: November 15, 2006 Published online: January 11, 2007 Roland S.G. Jones Department of Pharmacy and Pharmacology, University of Bath Claverton Down Bath, BA2 7AY (UK) Tel. +44 1225 383 935, Fax +44 1225 386 114, E-Mail [email protected] © 2006 S. Karger AG, Basel 1424–862X/06/0154–0202$23.50/0 Accessible online at: www.karger.com/nsg GABA B Receptors in the Entorhinal Cortex Neurosignals 2006–07;15:202–215 203 greater in layer II compared to layer V [7] . In a recent study we investigated whether this could be dependent on differences in modulation of release by presynaptic GABA B autoreceptors in the two layers [8] . Using the GABA B R agonist, baclofen, we found that these receptors depressed GABA release in the two layers to around the same extent. However, high concentrations of a GABA B R antagonist actually increased the release of GABA, suggesting a tonic feedback activation of the autoreceptor. The latter effect was restricted to layer V, but was of insufficient magnitude to fully explain the difference in frequency of sIPSCs recorded in the two layers. In addition to their postsynaptic actions and their role as autoreceptors, GABA B R have long been known to act as heteroreceptors, controlling the release of a wide variety of neuroactive agents, including amines, peptides and amino acids. In the present study we have examined the heteroreceptor control of glutamate release by GABA B R in the EC. There has been some debate over whether different subtypes of GABA B R mediate autoand heteroreceptor functions [9–14] . The development of more potent and specific GABA B R agonists and antagonists has allowed a more detailed examination of the possibility of receptor subtypes at distinct spatial locations. One agonist, CGP44533, has been suggested to be largely inactive at postsynaptic receptors [15] , whilst displaying a greater potency at heteroreceptors compared to autoreceptors [14, 16] . We have used this agonist to assess whether there may be differences in heteroand autoreceptors in the EC. The EC has been strongly implicated in the generation and propagation of temporal lobe epilepsies [see 4 ], and we have been looking at changes in control of transmitter release in chronically epileptic animals [e.g. 3 ]. It is apparent that mice that lack functional GABA B R, either by deletion of the GABA B1 subunit or mutation of the GABA B2 subunit, display a phenotype characterized by profound tonic-clonic seizure activity [17–19] . GABA B autoreceptor function has been shown to be decreased in the hippocampus [20, 21] after electrical kindling, and similar changes in heteroreceptors in both hippocam pus and amygdala have also been shown [22–24] . Currently, there is little information on whether presynaptic GABA B R are altered in the EC, although Gloveli et al. [25] reported an enhancement of autoreceptor function in layer III in kindled animals. Thus, in the second part of the present study, we examined the question of whether GABA B R control of either glutamate or GABA release is altered in rats with chronic pilocarpine-induced spontaneous seizures. Some of the results in this paper have been published in abstract form [26] . Methods Slice Preparation Experiments were performed in accordance with the UK Animals (Scientific Procedures) Act 1986, European Communities Council Directive 1986 (86/609/EEC) and the University of Bath ethical review document. Combined entorhinal-hippocampal slices were prepared from male Wistar rats, as previously described [27] . Rats were anaesthetised with an intramuscular injection of ketamine (120 mg/kg) plus xylazine (8 mg/kg) and decapitated. The brain was rapidly removed and immersed in oxygenated artificial cerebrospinal fluid (ACSF) chilled to 4 ° C. Slices (400
m) were cut using a Vibroslice, and stored in ACSF bubbled with 95% O 2 /5% CO 2 , at room temperature. Following recovery for at least 1 h, individual slices were transferred to a recording chamber mounted on the stage of an Olympus BX51 microscope. The chamber was perfused (2 ml/min) with oxygenated ACSF (pH 7.4) at 30–32 ° C. The ACSF contained (in m M ): NaCl (126), KCl (2.5), NaH 2 PO 4 (1.25), NaHCO 3 (24), MgSO 4 (2), CaCl 2 (2.5), and D -glucose (10). Neurones were visualized using differential interference contrast optics and an infrared video camera. Electrophysiological Recordings Patch pipettes (1–4 M ) were pulled from borosilicate glass on a Flaming/Brown microelectrode puller. For recording EPSCs, pipettes were filled with a solution containing (in m M ): Cs-methanosulphonate (120), HEPES (10), QX-314 (5), EGTA (10), CaCl 2 (0.34), NaCl (4), MgCl 2 (1), ATP-Na (4), and GTP-Na (0.4). IPSCs were recorded using a patch pipette solution containing: CsCl (120), HEPES (10), QX-314 (5), EGTA (10), CaCl 2 (0.34), MgCl 2 (1), ATP (4), and GTP (0.4). Solutions were adjusted to 290 mosm, and to pH 7.25–7.3 with CsOH. Whole-cell voltage-clamp recordings (holding potential –60 mV) were made from neurones in layer V and layer II of the medial division of the EC, using an Axopatch 200B amplifier. Series resistance compensation was not employed, but access resistance (10–30 M ) was monitored at regular intervals throughout each recording and neurones were discarded from analysis if it changed by more than 8 10%. Liquid junction potentials were estimated using the calculator of pClamp 8 software, and compensated for in the holding potentials. Under the above recording conditions, EPSCs in both layer II and V are mediated by the spontaneous release of glutamate acting at both AMPA and NMDA receptors [28] . No attempt was made to distinguish between events mediated by the two receptors, although pure NMDA receptor-mediated events are infrequent and most EPSCs reflect the activation of postsynaptic AMPA receptors. We did not pharmacologically block GABA A receptors in these recordings. However, using the internal and external solutions detailed above, all inward currents at the holding potential of –60 mV could be blocked by a combination of NBQX and 2-AP5 [29] . When recording IPSCs, the ACSF routinely contained NBQX (6-nitro-7-sulphamoylbenzo[f]quinoxal one-2,3-dione di-sodium; 20 M ) and 2-AP5 ( D,L -2-amino-5phosphonovalerate; 50 M ) to block AMPA and NMDA receptors, respectively. Under these recording conditions, i.e. with similar concentrations of Cl – intraand extracellularly and V h at –60 mV, IPSCs are recorded as inward currents, mediated by GABA acting at GABA A receptors [7, 8] . The inclusion of CsCl and QX314 in the patch pipette solutions ensured that in all studies, postsynaptic GABA B R were blocked in the recorded neurones. Spon-