Cellular Neurophysiology

Abstract

LE1 9HNRecent evidence suggests that principal neurones of the MNTB (medial nucleus of the trapezoid body) predominantly express GluRDo subunits (Geiger et al. 1995). Such Ca2+-permeable AMPA receptors exhibit rectification through a voltage-dependent block by internal polyamines (Kamboj et al. 1995). Previous reports of spermine-induced rectification of both native and cloned AMPA receptors have used agonist application. Here we set out to determine if intracellular spermine can induce rectification of a synaptic current in MNTB neurones.Transverse slices (200 ,um) of the brainstem were prepared from Lister Hooded rats (8-12 days), killed by decapitation. Whole-cell patch clamp recordings from MNTB neurones were made at 25°C with pipettes containing (mM): 130 CsCl, 10 Hepes, 5 EGTA and 1 MgCl2. Standard bicarbonatebuffered artificial cerebrospinal fluid with 1 mm Ca2+, 2 mM Mg2, 20-50 UM DL-AP5, 4-8 /M MK801, 5/M (5,7)-dichlorokynurenic acid and 2 /SM strychnine was continuously perfused over the slice. The presynaptic axons were stimulated with a bipolar electrode to evoke excitatory postsynaptic currents (EPSCs) in voltage clamped MNTB neurones.AMPA receptor-mediated EPSCs in MNTB neurones decay with a time course that can be described by the sum of two exponentials and have previously been found to have a linear current-voltage relationship. We have found that inclusion of spermine (100-500 uM) in the patch pipette resulted in rectification of the peak of the EPSC. Examination of the more slowly decaying component of the AMPA receptor-mediated EPSC suggests that it does not rectify. The rectification index at +60/-60 mV for the peak was 0 37 + 0-10 and for the slow component was 1-03 ± 0 33 (mean + S.D., n = 5 for 200 jM spermine). Studies on expressed channels have shown that argiotoxin 636 will block GluRDo subunits (Herlitze et al. 1993 We have identified some glutamate receptor subtypes expressed by magnocellular cholinergic neurones in dissociated culture. Dissociated cultures were prepared from postnatal day 11-14 Sprague-Dawley rats (killed by decapitated) using the method of Allen et al. (1993). After 5-30 days in culture, magnocellular neurones were voltageclamped using the whole-cell variant of the patch-clamp technique.Localized pressure application of 100 /M N-methyl-D-aspartate (NMDA) elicited an inward current that was sensitive to 1 mM Mg2+ and to the NMDA receptor antagonist 2-amino-5-phosphonopentanoate (AP5, 30 /M, n = 3). The non-NMDA receptor agonists a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA, 30 uM) and kainate (1 mM) elicited inward currents that were antagonized by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10-30 /M, n = 5 for each agonist).When applied by pressure ejection from a micropipette, the response to AMPA, but not to kainate, displayed strong desensitization. Bath application of AMPA and of kainate produced large steady-state currents with EC50 ± S.E.M. of 2 7 + 0A4 (n = 5) and 138 + 25 #M (n = 10), respectively. Cycloth...