Bidirectional synaptic plasticity in the cerebellum-like mammalian dorsal cochlear nucleus Kiyohiro Fujino* and Donata Oertel† Department of Physiology, University of Wisconsin, 1300 University Avenue, Madison, WI 53706 Edited by A. James Hudspeth, The Rockefeller University, New York, NY, and approved November 13, 2002 (received for review September 3, 2002) The dorsal cochlear nucleus integrates acoustic with multimodal depending on the solutions. Series resistance was compensated to sensory inputs from widespread areas of the brain. Multimodal Ͼ95%. Stimulus generation, data acquisition, and analyses were inputs are brought to spiny dendrites of fusiform and cartwheel performed with PCLAMP software (Axon Instruments). Cells were cells in the molecular layer by parallel fibers through synapses that held at Ϫ80 mV. Shocks (5- to 100-V amplitude, 100-␮s duration) are subject to long-term potentiation and long-term depression. were generated by a stimulator (Master-8, AMPI, Jerusalem) and Acoustic cues are brought to smooth dendrites of fusiform cells in delivered through a saline-filled glass pipette (3–5 ␮m in diameter) the deep layer by auditory nerve fibers through synapses that do to the molecular or deep layer between 100 and 200 ␮m from the not show plasticity. Plasticity requires Ca2؉-induced Ca2؉ release; recording site. Stimulus strength was set to evoke the largest its sensitivity to antagonists of N-methyl-D-aspartate and metabo- possible excitatory postsynaptic current (EPSC). EPSCs were mon- tropic glutamate receptors differs in fusiform and cartwheel cells. itored at 0.1 Hz. Cells’ input resistances were monitored by Ϫ10-mV voltage steps. Potentiation and depression were quantified uditory nerve fibers bring acoustic information to the as the ratio of the mean amplitude of EPSCs over a 5-min period Acochlear nucleus and feed it into several parallel pathways between 25 and 30 min or 55 and 60 min, referred to as 30 or 60 min that ascend through the brainstem. Pathways through the dorsal after the conditioning stimulus, respectively, and the mean ampli- cochlear nucleus (DCN) are thought to detect spectral cues for tude before the conditioning stimuli. Cells were excluded from Ͼ localizing sounds monaurally (1). Those through the ventral analysis if there was 20% change of input resistance or if the mean amplitudes of EPSCs for every minute during the control period cochlear nucleus (VCN) encode spectral and temporal charac- NEUROSCIENCE Ͼ teristics that allow sounds to be localized in the horizontal plane, varied by 10% from the overall mean amplitude during the through binaural circuits, and recognized. control period. In plots from individual cells (ordinates in pA), each Ϯ The DCN resembles the cerebellum, cartwheel cells being point represents the mean SE for six consecutive responses over homologues of cerebellar Purkinje cells (2). Fusiform cells 1 min. Plots of normalized responses (ordinate is %EPSC) show Ϯ project to the inferior colliculus. Granule cells in the vicinity of mean SE of responses from different cells. Numerical data are Ϯ the cochlear nuclei are innervated by diverse regions of the presented as mean SD with number of cells tested. brain, including the dorsal column nuclei (1), vestibular periph- In most experiments, the pipettes contained (in mM) 113 ery (3), and auditory nuclei (4, 5). Their unmyelinated axons, K-gluconate, 4.5 MgCl2, 14 Tris2-phosphocreatine, 9 Hepes, 0.1 parallel fibers, contact spiny dendrites of fusiform and cartwheel EGTA, 4 Na2-ATP, and 0.3 Tris-GTP (pH adjusted to 7.25 with cells. Myelinated auditory nerve fibers contact smooth basal KOH). In some experiments, 0.1% biocytin was added to the dendrites of fusiform cells. The DCN resembles the electrosen- pipette. In experiments in which the intracellular calcium con- sory lobes in fishes even more closely (6, 7). centration was strongly buffered, pipettes contained (in mM) 108 In cerebellar circuits, the gain of one of two converging K-gluconate, 4.5 MgCl2, 14 Tris2-phosphocreatine, 9 Hepes, 9 systems of inputs varies as a function of activity. Long-term EGTA, 4 Na2-ATP, and 0.3 Tris-GTP (pH 7.25). Recordings in depression (LTD) at parallel fiber synapses in cerebellar Pur- Fig. 1 A–C were made with pipettes filled with a CsCl-based ͞ ͞ ͞ ͞ ͞ kinje cells evoked by coincident excitation through parallel fiber solution (in mM): 140 CsCl 1 KCl 1 NaCl 3 MgCl2 9 Hepes 10 ͞ ͞ and climbing fiber synapses plays a role in coordination and EGTA 2 QX-314 0.1% biocytin; pH was adjusted to 7.25 with motor learning (8). Long-term potentiation (LTP) and LTD at CsOH. Ryanodine and thapsigargin (Calbiochem) were dis- parallel fiber synapses are evoked in electric fishes by convergent solved in DMSO, resulting in a 0.1% concentration of DMSO in excitation by parallel fibers and sensory afferents to compensate the pipette. Ten millimolar caffeine replaced 5 mM K-gluconate for an animal’s own signals in localizing external sources (9). We in the pipette solution. show that principal cells of the DCN combine multimodal inputs The normal external saline contained (in mM): 130 NaCl, 3 KCl, through parallel fibers whose strength is modulated by activity 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, 20 NaHCO3, 3 Hepes, and 10 ͞ with invariant acoustic input through auditory nerve fibers. glucose; saturated with 95% O2 5% CO2; pH adjusted to 7.4 with NaOH. To prevent precipitation, LY341495 (Tocris) was added to ͞ ͞ ͞ Methods a Hepes-buffered saline (in mM): 138 NaCl 4.2 KCl 2.4 CaCl2 1.3 ͞ ͞ Coronal slices (200 ␮m) containing the DCN were prepared MgCl2 10 Hepes 10 glucose; saturated with 100% O2;pH7.4. from ICR mice (Harlan–Sprague–Dawley) between 18 and 20 Glycinergic and GABAAergic inhibitory inputs were routinely ␮ ␮ days old with an oscillating tissue slicer (VT1000S, Leica Mi- blocked by 1 M strychnine and 50 M picrotoxin. DNQX crosystems, Nussloch, Germany). Slices were held in a 300-␮l (6,7-dinitroquinoxaline-2,3-dione), APV (2-amino-5-phosphono- chamber that was superfused at 3–5 ml͞min with oxygenated valeric acid), transACPD (t-ACPD, Tocris, Ellisville, MO), and saline at Ϸ33°C. Experiments were done in accordance with the protocols and guidelines of the Animal Care and Use Committee at the University of Wisconsin, Madison. This paper was submitted directly (Track II) to the PNAS office. Whole-cell patch-clamp recordings were generally made with 3- Abbreviations: APV, 2-amino-5-phosphonovaleric acid; DCN, dorsal cochlear nucleus; ⍀ DNQX, 6,7-dinitroquinoxaline-2,3-dione; EPSC, excitatory postsynaptic current; LTD, long- to 5-M pipettes filled with a K-gluconate-based solution. Voltages term depression; LTP, long-term potentiation; mGluR, metabotropic glutamate receptor; and currents were measured through an Axopatch 200B amplifier, NMDA, N-methyl-D-aspartate. low-pass filtered at 5 kHz and digitized at 16.7–25 kHz through a *Present address: Department of Otolaryngology–Head and Neck Surgery, Graduate Digidata 1200 interface (Axon Instruments, Foster City, CA). School of Medicine, Kyoto University, Kyoto 606-8507, Japan. Voltages were corrected for junction potentials, Ϫ5toϪ12 mV †To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0135345100 PNAS ͉ January 7, 2003 ͉ vol. 100 ͉ no. 1 ͉ 265–270 Downloaded by guest on September 30, 2021 tification of cells was based on electrophysiological criteria developed previously (10–12). Depolarizing current evoked trains of uniform action potentials in fusiform and complex action potentials in cartwheel cells. The resting potentials were Ϫ58.6 Ϯ 4.1 mV in fusiform cells (n ϭ 195) and Ϫ64.6 Ϯ 11.1 mV in cartwheel cells (n ϭ 181). Three excitatory synaptic responses were compared: (i) par- allel fiber inputs to apical dendrites of fusiform cells, (ii) auditory nerve inputs to basal dendrites of fusiform cells, and (iii) parallel fiber inputs to cartwheel cells. The deep layer contains axons from two possible sources of excitation, auditory nerve fibers and T stellate cells (13). For simplicity, we will attribute all responses to stimulation in the deep layer to auditory nerve fibers because no heterogeneity was detected. EPSCs were mediated through N-methyl-D-aspartate (NMDA) and AMPA receptors. Fifty-micromolar APV, an antagonist of NMDA receptors, decreased the amplitude and duration of synaptic currents measured at depolarized potentials. The APV- insensitive currents were blocked by 20 ␮M DNQX, indicating that they are mediated through AMPA receptors (Fig. 1 A–C). TransACPD (t-ACPD), an agonist for group 1 and 2 metabo- tropic glutamate receptors (mGluRs) (14), and L-AP4, an agonist for group 3 mGluRs (15), were used to test for the contribution of mGluRs to synaptic transmission between par- allel fibers and their fusiform and cartwheel cell targets (14, 16, 17). Applications were made while the holding current and the amplitude of EPSCs evoked by stimulation of parallel fibers were monitored. Dose–response relationships for t-ACPD were sig- moid, saturating at Ϸ100 ␮M. At 200 ␮M, t-ACPD shifted the holding current inwardly in fusiform (70.3 Ϯ 20.1 pA, n ϭ 10; Fig. Fig. 1. Glutamatergic receptors of the AMPA, NMDA, and mGluR subtypes 1D) and cartwheel cells (67.5 Ϯ 20.9 pA, n ϭ 8; Fig. 1F). In contribute to synaptic responses in fusiform and cartwheel cells. (A)Ina cartwheel cells, t-ACPD also decreased the amplitude of the fusiform cell, EPSCs were evoked by stimulating the molecular layer while the Ϯ ϭ Ϫ ϩ EPSCs (32.1 6.1% decrease, n 8; Fig. 1F). The actions of holding potential of the cell was varied between 80 and 40 mV in 20-mV ␮ steps. A slow component that was prominent at depolarizing holding poten- t-ACPD had an EC50, when fit with a Hill function, near 25 M tials was blocked by 50 ␮M APV, indicating that it was mediated through for all these effects (fusiform holding current 28.3 ␮M; cartwheel NMDA receptors.