Tetsuhiro Tsujimoto

Presynaptic Calcium Current Modulation by a Metabotropic Glutamate Receptor

Metabotropic glutamate receptors (mGluRs) regulate transmitter release at mammalian central synapses. However, because of the difficulty of recording from mammalian presynaptic terminals, the mechanism underlying mGluR-mediated presynaptic inhibition is not known. Here, simultaneous recordings from a giant presynaptic terminal, the calyx of Held, and its postsynaptic target in the medial nucleus of the trapezoid body were obtained in rat brainstem slices. Agonists of mGluRs suppressed a high voltage-activated P/Q-type calcium conductance in the presynaptic terminal, thereby inhibiting transmitter release at this glutamatergic synapse. Because several forms of presynaptic modulation and plasticity are mediated by mGluRs, this identification of a target ion channel is a first step toward elucidation of their molecular mechanism.

INACTIVATION OF PRESYNAPTIC CALCIUM CURRENT CONTRIBUTES TO SYNAPTIC DEPRESSION AT A FAST CENTRAL SYNAPSE

Voltage-gated calcium channels are well characterized at neuronal somata but less thoroughly understood at the presynaptic terminal where they trigger transmitter release. In order to elucidate how the intrinsic properties of presynaptic calcium channels influence synaptic function, we have made direct recordings of the presynaptic calcium current (I(pCa)) in a brainstem giant synapse called the calyx of Held. The current was pharmacologically classified as P-type and exhibited marked inactivation. The inactivation was largely dependent upon the inward calcium current magnitude rather than the membrane potential, displayed little selectivity between divalent charge carriers (Ca2+, Ba2+ and Sr+), and exhibited slow recovery. Simultaneous pre- and postsynaptic whole-cell recording revealed that I(pCa) inactivation predominantly contributes to posttetanic depression of EPSCs. Thus, because of its slow recovery, I(pCa) inactivation underlies this short-term synaptic plasticity.

Facilitation of the presynaptic calcium current at an auditory synapse in rat brainstem

1 The presynaptic calcium current (IpCa) was recorded from the calyx of Held in rat brainstem slices using the whole‐cell patch clamp technique. 2 Tetanic activation of IpCa by 1 ms depolarizing voltage steps markedly enhanced the amplitude of IpCa. Using a paired pulse protocol, the second (test) response was facilitated with inter‐pulse intervals of less than 100 ms. The facilitation was greater at shorter intervals and was maximal (about 20 %) at intervals of 5–10 ms. 3 When the test pulse duration was extended, the facilitation was revealed as an increased rate of IpCa activation. From the current‐voltage relationship measured at 1 ms from onset, facilitation could be described by a shift in the half‐activation voltage of about −4 mV. 4 I pCa facilitation was not attenuated when guanosine‐5′‐O‐(3‐thiotriphosphate) (GTPγS) or guanosine‐5′‐O‐(2‐thiodiphosphate) (GDPβS) was included in the patch pipette, suggesting that G‐proteins are not involved in this phenomenon. 5 On reducing [Ca2+]o, the magnitude of facilitation diminished proportionally to the amplitude of IpCa. Replacement of [Ca2+]o by Ba2+ or Na+, or buffering of [Ca2+]i with EGTA or BAPTA attenuated IpCa facilitation. 6 We conclude that repetitive presynaptic activity can facilitate the presynaptic Ca2+ current through a Ca2+‐dependent mechanism. This mechanism would be complementary to the action of residual Ca2+ on the exocytotic machinery in producing activity‐dependent facilitation of synaptic responses.

Voltage-activated Ca2+ channels and their role in the endocrine function of the pituitary gland in newborn and adult mice

We have prepared fresh pituitary gland slices from adult and, for the first time, from newborn mice to assess modulation of secretory activity via voltage‐activated Ca2+ channels (VACCs). Currents through VACCs and membrane capacitance have been measured with the whole‐cell patch‐clamp technique. Melanotrophs in newborns were significantly larger than in adults. In both newborn and adult melanotrophs activation of VACCs triggered exocytosis. All pharmacologically isolated VACC types contributed equally to the secretory activity. However, the relative proportion of VACCs differed between newborns and adults. In newborn cells L‐type channels dominated and, in addition, an exclusive expression of a toxin‐resistant R‐type‐like current was found. The expression of L‐type VACCs was up‐regulated by the increased oestrogen levels observed in females, and was even more emphasized in the cells of pregnant females and oestrogen‐treated adult male mice. We suggest a general mechanism modulating endocrine secretion in the presence of oestrogen and particularly higher sensitivity to treatments with L‐type channel blockers during high oestrogen physiological states.

313 Inhibitory synaptic transmission in mice lacking synapsin I

Akira Yoshida’ , Mica Imaizumi2, Kounosuke Kumakura2, Tohru Yoshioka’ In the presence of inositolB-phosphate, we determined an interaction of synaptotagmin and syntaxin by BIAcore system. The carboxy-terminal region of recombinant syntaxin was immobilized on a sensor chip surface, and the cytoplasmic region of recombinant synaptotagmin was injected into the system with 0.5 mM of CaClz and various concentration of inositol-6-phosphate. The binding activity of synaptotagmin to syntaxin was partially inhibited at 100nM of inositoi-6-phosphate. The dissociation phase of synaptotagmin from syntaxin in the presence of high concentration of CaClz was constituted by two components which showed fast and slow dissociation rate constant respectively. The inositol-gphosphate reduced the slower component in the dessociateion phase, therefore the inositol polyphosphate attenuated the apparent binding activity of synaptotagmin to syntaxin

317 Inactivation of presynaptic calcium current underlying synaptic depression

Hiromu Yawo’, Shuichi Saheki2 Norepinephrine (NE) potentiated the transmitter release from the giant presynaptic terminal of chick ciliary ganglion through a receptor pharmacologically different from alphaand beta-adrenergic receptors (1) which may’activate cGMP-protein kinase G cascade. The mechanism how NE activates guanylyl cyclase was investigated. The NEdependent potentiation was not affected by either NO-synthase inhibitor, N”nitro-L-arginine methyl ester (L-NAME, 100microM) or NO-scavenger, hemoglobin (30microM). Moreover, the NO-donors, neither sodium nitroprusside (100microM) nor (&)-(E)-4-Methyl-P-[(E)-hydroxyiminol-5-nitro-8methoxyS-hexenamide (NORl, POmicroM) did not potentiate the transmitter release even in the presence of phosphodiesterase inhibitor, 3-isobutyl-1-methyxanthine (IBMX). Therefore, the NO-sensitive soluble guanylyl cyclase appears not to be expressed in the giant presynaptic terminals of embryonic chick ciliary ganglion. It is concluded that the novel adrenergic receptor different from both alphaand beta-adrenergic receptors activate guanylyl cyclase without involving NO. 1. Yawo, H. (1996) J. Physiol. Lond. 493:385-391.