Brian Billups

Presynaptic Rat Kv1.2 Channels Suppress Synaptic Terminal Hyperexcitability Following Action Potential Invasion

Voltage‐gated K+ channels activating close to resting membrane potentials are widely expressed and differentially located in axons, presynaptic terminals and cell bodies. There is extensive evidence for localisation of Kv1 subunits at many central synaptic terminals but few clues to their presynaptic function. We have used the calyx of Held to investigate the role of presynaptic Kv1 channels in the rat by selectively blocking Kv1.1 and Kv1.2 containing channels with dendrotoxin‐K (DTX‐K) and tityustoxin‐Kα (TsTX‐Kα) respectively. We show that Kv1.2 homomers are responsible for two‐thirds of presynaptic low threshold current, whilst Kv1.1/Kv1.2 heteromers contribute the remaining current. These channels are located in the transition zone between the axon and synaptic terminal, contrasting with the high threshold K+ channel subunit Kv3.1 which is located on the synaptic terminal itself. Kv1 homomers were absent from bushy cell somata (from which the calyx axons arise); instead somatic low threshold channels consisted of heteromers containing Kv1.1, Kv1.2 and Kv1.6 subunits. Current‐clamp recording from the calyx showed that each presynaptic action potential (AP) was followed by a depolarising after‐potential (DAP) lasting around 50 ms. Kv1.1/Kv1.2 heteromers had little influence on terminal excitability, since DTX‐K did not alter AP firing. However TsTX‐Kα increased DAP amplitude, bringing the terminal closer to threshold for generating an additional AP. Paired pre‐ and postsynaptic recordings confirmed that this aberrant AP evoked an excitatory postsynaptic current (EPSC). We conclude that Kv1.2 channels have a general presynaptic function in suppressing terminal hyperexcitability during the depolarising after‐potential.

Unmasking group III metabotropic glutamate autoreceptor function at excitatory synapses in the rat CNS

Presynaptic group III metabotropic glutamate receptor (mGluR) activation by exogenous agonists (such as l‐2‐amino‐4‐phosphonobutyrate (l‐AP4)) potently inhibit transmitter release, but their autoreceptor function has been questioned because endogenous activation during high‐frequency stimulation appears to have little impact on synaptic amplitude. We resolve this ambiguity by studying endogenous activation of mGluRs during trains of high‐frequency synaptic stimuli at the calyx of Held. In vitro whole‐cell patch recordings were made from medial nucleus of the trapezoid body (MNTB) neurones during 1 s excitatory postsynaptic current (EPSC) trains delivered at 200 Hz and at 37°C. The group III mGluR antagonist (R,S)‐cyclopropyl‐4‐phosphonophenylglycine (CPPG, 300 μm) had no effect on EPSC short‐term depression, but accelerated subsequent recovery time course (τ: 4.6 ± 0.8 s to 2.4 ± 0.4 s, P= 0.02), and decreased paired pulse ratio from 1.18 ± 0.06 to 0.97 ± 0.03 (P= 0.01), indicating that mGluR activation reduced release probability (P). Modelling autoreceptor activation during repetitive stimulation revealed that as P declines, the readily releasable pool size (N) increases so that the net EPSC (NP) is unchanged and short‐term depression proceeds with the same overall time course as in the absence of autoreceptor activation. Thus, autoreceptor action on the synaptic response is masked but the synapse is now in a different state (lower P, higher N). While vesicle replenishment clearly underlies much of the recovery from short‐term depression, our results show that the recovery time course of P also contributes to the reduced response amplitude for 1–2 s. The results show that passive equilibration between N and P masks autoreceptor modulation of the EPSC and suggests that mGluR autoreceptors function to change the synaptic state and distribute metabolic demand, rather than to depress synaptic amplitude.

Unmasking group III metabotropic glutamate autoreceptor function at excitatory synapses

Presynaptic group III metabotropic glutamate receptor (mGluR) activation by exogenous agonists (such as l‐2‐amino‐4‐phosphonobutyrate (l‐AP4)) potently inhibit transmitter release, but their autoreceptor function has been questioned because endogenous activation during high‐frequency stimulation appears to have little impact on synaptic amplitude. We resolve this ambiguity by studying endogenous activation of mGluRs during trains of high‐frequency synaptic stimuli at the calyx of Held. In vitro whole‐cell patch recordings were made from medial nucleus of the trapezoid body (MNTB) neurones during 1 s excitatory postsynaptic current (EPSC) trains delivered at 200 Hz and at 37°C. The group III mGluR antagonist (R,S)‐cyclopropyl‐4‐phosphonophenylglycine (CPPG, 300 μm) had no effect on EPSC short‐term depression, but accelerated subsequent recovery time course (τ: 4.6 ± 0.8 s to 2.4 ± 0.4 s, P= 0.02), and decreased paired pulse ratio from 1.18 ± 0.06 to 0.97 ± 0.03 (P= 0.01), indicating that mGluR activation reduced release probability (P). Modelling autoreceptor activation during repetitive stimulation revealed that as P declines, the readily releasable pool size (N) increases so that the net EPSC (NP) is unchanged and short‐term depression proceeds with the same overall time course as in the absence of autoreceptor activation. Thus, autoreceptor action on the synaptic response is masked but the synapse is now in a different state (lower P, higher N). While vesicle replenishment clearly underlies much of the recovery from short‐term depression, our results show that the recovery time course of P also contributes to the reduced response amplitude for 1–2 s. The results show that passive equilibration between N and P masks autoreceptor modulation of the EPSC and suggests that mGluR autoreceptors function to change the synaptic state and distribute metabolic demand, rather than to depress synaptic amplitude.

Colocalization of vesicular glutamate transporters in the rat superior olivary complex

Vesicular glutamate transporters (VGLUTs) are responsible for the accumulation of the excitatory neurotransmitter glutamate into synaptic vesicles. It is currently controversial whether the two isoforms found in glutamatergic neurons, VGLUT1 and VGLUT2, are present at the same synapse or have entirely complementary patterns of distribution. Using fluorescent immunohistochemistry, this study examines the colocalization of these two transporters in the rat superior olivary complex (SOC) between postnatal day (P) 5 and 29. The medial and lateral superior olives (MSO; LSO) stain for both VGLUT1 and VGLUT2 at all ages studied, with VGLUT1 levels doubling over this developmental period and VGLUT2 levels remaining unchanged. The ventral nucleus of the trapezoid body (VNTB) strongly labels only for VGLUT2, despite the fact that glutamatergic synapses are present that are formed from collaterals of axons that go on to form synapses containing both VGLUT1 and VGLUT2. Principal neurons of the medial nucleus of the trapezoid body (MNTB) are surrounded by the calyx of Held presynaptic terminal, which is large enough to allow examination of VGLUT localization within a synapse. Throughout its postnatal developmental period a single calyx synapse contains both VGLUT1 and VGLUT2. Whereas VGLUT1 levels are greatly up-regulated from P5 to P29, VGLUT2 levels remain high. As the abundance of VGLUT determines the quantal size, this up-regulation will increase excitatory postsynaptic currents (EPSCs) and have influences on synaptic physiology.

Non-calyceal excitatory inputs mediate low fidelity synaptic transmission in rat auditory brainstem slices

Principal neurons of the medial nucleus of the trapezoid body (MNTB) receive a synaptic input from a single giant calyx terminal that generates a fast‐rising, large excitatory postsynaptic current (EPSC), each of which are supra‐threshold for postsynaptic action potential generation. Here, we present evidence that MNTB principal neurons receive multiple excitatory synaptic inputs generating slow‐rising, small EPSCs that are also capable of triggering postsynaptic action potentials but are of non‐calyceal origin. Both calyceal and non‐calyceal EPSCs are mediated by α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA) and N‐methyl‐d‐aspartate (NMDA) receptor activation; however, the NMDA receptor‐mediated response is proportionally larger at the non‐calyceal synapses. Non‐calyceal synapses generate action potentials in MNTB principal neurons with a longer latency and a lower reliability than the large calyceal input. They constitute an alternative low fidelity synaptic input to the fast and secure relay transmission via the calyx of Held synapse.

Detecting synaptic connections in the medial nucleus of the trapezoid body using calcium imaging

Abstract. The study of synaptic transmission in brain slices generally entails the patch-clamping of postsynaptic neurones and stimulation of identified presynaptic axons using a remote electrical stimulating electrode. Although patch recording from postsynaptic neurones is routine, many presynaptic axons take tortuous turns and are severed in the slicing procedure, blocking propagation of the action potential to the synaptic terminal and preventing synaptic stimulation. Here we demonstrate a method of using calcium imaging to select postsynaptic cells with functional synaptic inputs prior to patch-clamp recording. We have used this method for exploring transmission in the auditory brainstem at the medial nucleus of the trapezoid body neurones, which are innervated by axons from the contralateral cochlear nucleus. Brainstem slices were briefly loaded with the calcium indicator fura-2 AM and stimulated with an electrode placed on the midline. Electrical stimulation caused a rise in intracellular calcium concentration in those postsynaptic neurones with active synaptic connections. Since <10% of the medial nucleus of the trapezoid body neurones retain viable synaptic inputs following the slicing procedure, preselecting those cells with active synapses dramatically increased our recording success. This detection method will greatly ease the study of synaptic responses in brain areas where suprathreshold synaptic inputs occur but connectivity is sparse.