Effect of VGLUT inhibitors on glutamatergic synaptic transmission in the rodent hippocampus and prefrontal cortex

doi:10.1016/j.neuint.2013.10.001

Neurochemistry International

Volume 73, July 2014, Pages 159-165

https://doi.org/10.1016/j.neuint.2013.10.001 Get rights and content

Highlights

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Inhibition of VGLUTs results in depression of glutamatergic synaptic transmission.
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The endogenous VGLUT inhibitor Xanthurenic Acid has similar actions.
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VGLUT inhibition modulates NMDA-R mediated transmission in the prefrontal cortex.
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This suggests VGLUT modulation may be a target for development of new therapeutics.

Abstract

Vesicular glutamate transporters (VGLUTs) are known to be important in the uptake of glutamate into vesicles in the presynaptic terminal; thereby playing a role in synaptic function. VGLUT dysfunction has also been suggested in neurological and psychiatric disorders such as epilepsy and schizophrenia. A number of compounds have been identified as VGLUT inhibitors; however, little is known as to how these compounds affect synaptic transmission. We therefore investigated the effects of structurally unrelated VGLUT inhibitors on synaptic transmission in the rodent hippocampus and prefrontal cortex.

In the CA1 and dentate gyrus regions of the in vitro slice preparation of mouse hippocampus, AMPA receptor-mediated field excitatory postsynaptic potentials (fEPSPs) were evoked in response to Schaffer collateral/commissural pathway stimulation. Application of the VGLUT inhibitors Rose Bengal (RB), Congo Red (CR) or Chicago Sky Blue 6B (CB) resulted in a concentration-related reduction of fEPSP amplitudes. RB (30 μM) or CB (300 μM) also depressed NMDA receptor-mediated responses in the CA1 region. The naturally occurring kynurenine Xanthurenic Acid (XA) is reported to be a VGLUT inhibitor. We found XA attenuated both AMPA and NMDA receptor-mediated synaptic transmission. The potency order of the VGLUT inhibitors was consistent with literature K_i values for VGLUT inhibition.

Impaired glutamatergic neurotransmission is believed to contribute to schizophrenia, and VGLUTs have also been implicated in this disease. We therefore investigated the effect of VGLUT inhibition in the prefrontal cortex. Application of the VGLUT inhibitors RB or CB resulted in a concentration-dependent reduction in the amplitude of glutamate receptor-mediated fEPSPs recorded in layer V/VI in response to stimulation in the forceps minor.

We conclude that VGLUT inhibitors can modulate glutamatergic synaptic transmission in the PFC and hippocampus. This could be important in the pathophysiology of nervous system disorders and might represent a target for developing novel pharmacological therapies.

Introduction

Glutamate is the principal excitatory neurotransmitter in the central nervous system (CNS) and the mechanisms underlying glutamatergic neurotransmission are of considerable interest for our understanding of normal synaptic function and pathophysiology. Glutamate is stored in vesicles located in the presynaptic terminal and is released into the synaptic cleft following fusion of a vesicle with the cell membrane (Sudhof, 1995, Takamori, 2006). In the presynaptic terminal, the principal role of the vesicular glutamate transporters (VGLUTs) is to load glutamate into these vesicles via a proton-dependent electrochemical gradient (Takamori, 2006, Thompson et al., 2005). In addition, VGLUTs may also affect synaptic vesicle clustering and mobility in the presynaptic terminal (Fremeau et al., 2004, Shigeri et al., 2004, Siksou et al., 2013, Takamori, 2006).

Three isoforms of VGLUT have been identified and these belong to the type I phosphate transporter (SLC17) family. VGLUT1 was first cloned in 1994 (Ni et al., 1994) and was initially identified as brain-specific Na⁺-dependent inorganic phosphate transporter 1 (BNP1), but was renamed due to expression on the vesicle membrane and recognition of its ability to transport glutamate (Bellocchio et al., 2000, Takamori et al., 2000, Takamori, 2006). The two other isoforms, VGLUT2 and VGLUT3, were subsequently identified in the early 2000s (see (Takamori, 2006 and references therein). Consistent with a role in glutamatergic synaptic transmission, VGLUT 1 and 2 are expressed in glutamatergic terminals, with little overlap in expression (Fremeau et al., 2001, Fremeau et al., 2004, Fujiyama et al., 2001), although in some instances they may be co-expressed (Herzog et al., 2006). VGLUT3 may also have a role in glutamatergic transmission (Higley et al., 2011), but expression in neurones expressing other neurotransmitters, extrasynaptic expression and expression in peripheral tissues, such as the liver, suggests additional roles (Shigeri et al., 2004, Takamori, 2006).

VGLUTs play a role in maintaining normal synaptic function as demonstrated in studies in mice where transporters are either artificially expressed or are knocked out. Introduction of VGLUT1 into GABA-expressing neurones results in the cells producing glutamatergic autapses (Takamori et al., 2000), and in mice lacking VGLUT there are impairments of synaptic transmission, sensory processing, coordination and learning and memory (Balschun et al., 2010, Fremeau et al., 2004, Moechars et al., 2006, Seal et al., 2008, Seal et al., 2009, Smear et al., 2007, Wojcik et al., 2004). In addition to a role in normal synaptic activity, there is also evidence that modulation of VGLUT function or expression may play a role in neurological and psychiatric diseases including: epilepsy (Juge et al., 2010, Schallier et al., 2009), pain (Moechars et al., 2006, Seal et al., 2009) and schizophrenia (Eastwood and Harrison, 2005, Varea et al., 2012, Uezato et al., 2009, Oni-Orisan et al., 2008).

A number of compounds have been identified as VGLUT inhibitors (Pietrancosta et al., 2010, Shigeri et al., 2004, Thompson et al., 2005), and there is some evidence that these may modulate neuronal function in vivo (He et al., 2013). It is, however, perhaps surprising that there is little data regarding the action of these compounds on synaptic transmission. Recently, we demonstrated an effect of VGLUT inhibition on synaptic transmission in the mouse DG (Neale et al., 2013). In the current study we expand on those studies and test the action of structurally unrelated VGLUT inhibitors, Rose Bengal, Congo Red or Chicago Sky Blue 6B (Fig. 1) and the naturally occurring kynurenine and VGLUT inhibitor XA (Bartlett et al., 1998, Carrigan et al., 2002, Schwarcz et al., 2012), on glutamate receptor-mediated synaptic transmission in the prefrontal cortex and hippocampal CA1 and DG regions of the mouse.

Section snippets

Slice preparation

Adult (>4 weeks) female C57Bl6/J mice (Harlan, UK) were killed by decapitation and the brain was removed and placed into ice-cold oxygenated sucrose Krebs’ medium containing (mM): sucrose 202, KCl 2, KH₂PO₄ 1.25, MgSO₄ 10, CaCl₂ 0.5, NaHCO₃ 26, glucose 10. To record from the prefrontal cortex, 400 μm coronal slices were prepared, and for hippocampal recording 300 μm parasagittal slices were prepared with an oscillating microtome (Integraslice; Campden Instruments Ltd., Loughborough, UK). Slices

Effect of VGLUT inhibitors on transmission in the dentate gyrus region of the hippocampus

In the DG, stimuli evoked fEPSPs which were abolished in the presence of 10 μM of the AMPA receptor antagonist NBQX (data not shown; Sheardown et al., 1990). When pairs of stimuli were applied (40 ms interval), fEPSPs exhibited paired-pulse depression, indicative of medial perforant pathway activation (Macek et al., 1996). The VGLUT inhibitors Rose Bengal (1–30 μM), Congo Red (100 μM) and Chicago Sky Blue 6B (100 and 300 μM) decreased the amplitude of the fEPSP recorded in the DG (Fig. 2). In the

Discussion

In the present study we have characterised the actions of VGLUT inhibitors on glutamate receptor-mediated synaptic transmission in the prefrontal cortex and hippocampal CA1 and DG regions of the mouse and find evidence of VGLUT inhibition depressing neurotransmission in both regions. We have also characterised the action of XA, a naturally occurring VGLUT inhibitor in the CNS, (Gobaille et al., 2008, Schwarcz et al., 2012), and find this depresses both AMPA and NMDA receptor-mediated synaptic

References (51)

S.K. Ahmed et al.
Use of the hydantoin isostere to produce inhibitors showing selectivity toward the vesicular glutamate transporter versus the obligate exchange transporter system x(c)(-)
Bioorg. Med. Chem. Lett.
(2011)
R.D. Bartlett et al.
Substituted quinolines as inhibitors of l-glutamate transport into synaptic vesicles
Neuropharmacology
(1998)
S.L. Eastwood et al.
Decreased expression of vesicular glutamate transporter 1 and complexin II mRNAs in schizophrenia: further evidence for a synaptic pathology affecting glutamate neurons
Schizophr. Res.
(2005)
J.L. Etoga et al.
Conformationally-restricted amino acid analogues bearing a distal sulfonic acid show selective inhibition of system x(c)(-) over the vesicular glutamate transporter
Bioorg. Med. Chem. Lett.
(2010)
R.T. Fremeau et al.
The expression of vesicular glutamate transporters defines two classes of excitatory synapse
Neuron
(2001)
P. Frid et al.
Congo red and protein aggregation in neurodegenerative diseases
Brain Res. Rev.
(2007)
Z. He et al.
Chicago sky blue 6B, a vesicular glutamate transporters inhibitor, attenuates methamphetamine-induced hyperactivity and behavioral sensitization in mice
Behav. Brain Res.
(2013)
N. Juge et al.
Metabolic control of vesicular glutamate transport and release
Neuron
(2010)
J.N. Kew et al.
Differential regulation of synaptic transmission by mGlu2 and mGlu3 at the perforant path inputs to the dentate gyrus and CA1 revealed in mGlu2 −/− mice
Neuropharmacology
(2002)
A. Oni-Orisan et al.
Altered vesicular glutamate transporter expression in the anterior cingulate cortex in schizophrenia
Biol. Psychiatry
(2008)

N. Pietrancosta et al.

Rose Bengal analogs and vesicular glutamate transporters (VGLUTs)

Bioorg. Med. Chem.

(2010)

A. Schallier et al.

VGLUT2 heterozygous mice show more susceptibility to clonic seizures induced by pentylenetetrazol

Neurochem. Int.

(2009)

R.P. Seal et al.

Sensorineural deafness and seizures in mice lacking vesicular glutamate transporter 3

Neuron

(2008)

Y. Shigeri et al.

Molecular pharmacology of glutamate transporters, EAATs and VGLUTs

Brain Res. Brain Res. Rev.

(2004)

M.C. Smear et al.

Vesicular glutamate transport at a central synapse limits the acuity of visual perception in zebrafish

Neuron

(2007)

S. Takamori

VGLUTs: ‘exciting’ times for glutamatergic research?

Neurosci. Res.

(2006)

E. Varea et al.

Expression of PSA-NCAM and synaptic proteins in the amygdala of psychiatric disorder patients

J. Psychiatr. Res.

(2012)

P. Anderson et al.

The Hippocampus Book

(2007)

D. Balschun et al.

Vesicular glutamate transporter VGLUT1 has a role in hippocampal long-term potentiation. and spatial reversal learning

Cereb. Cortex

(2010)

E.E. Bellocchio et al.

Uptake of glutamate into synaptic vesicles by an inorganic phosphate transporter

Science

(2000)

C.A. Benassi et al.

Tryptophan metabolism in schizophrenic patients

J. Neurochem.

(1961)

A.J. Berger et al.

Glycine uptake governs glycine site occupancy at NMDA receptors of excitatory synapses

J. Neurophysiol.

(1998)

C.N. Carrigan et al.

Synthesis and in vitro pharmacology of substituted quinoline-2,4-dicarboxylic acids as inhibitors of vesicular glutamate transport

J. Med. Chem.

(2002)

R.T. Fremeau et al.

Vesicular glutamate transporters 1 and 2 target to functionally distinct synaptic release sites

Science

(2004)

F. Fujiyama et al.

Immunocytochemical localization of candidates for vesicular glutamate transporters in the rat cerebral cortex

J. Comp. Neurol.

(2001)

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Effect of VGLUT inhibitors on glutamatergic synaptic transmission in the rodent hippocampus and prefrontal cortex

Highlights

Abstract

Introduction

Section snippets

Slice preparation

Effect of VGLUT inhibitors on transmission in the dentate gyrus region of the hippocampus

Discussion

Bioorg. Med. Chem. Lett.

Neuropharmacology

Schizophr. Res.

Bioorg. Med. Chem. Lett.

Neuron

Brain Res. Rev.

Behav. Brain Res.

Neuron

Neuropharmacology

Biol. Psychiatry

Bioorg. Med. Chem.

Neurochem. Int.

Neuron

Brain Res. Brain Res. Rev.

Neuron

Neurosci. Res.

J. Psychiatr. Res.

The Hippocampus Book

Vesicular glutamate transporter VGLUT1 has a role in hippocampal long-term potentiation. and spatial reversal learning

Cereb. Cortex

Uptake of glutamate into synaptic vesicles by an inorganic phosphate transporter

Science

Tryptophan metabolism in schizophrenic patients

J. Neurochem.

Glycine uptake governs glycine site occupancy at NMDA receptors of excitatory synapses

J. Neurophysiol.

Synthesis and in vitro pharmacology of substituted quinoline-2,4-dicarboxylic acids as inhibitors of vesicular glutamate transport

J. Med. Chem.

Vesicular glutamate transporters 1 and 2 target to functionally distinct synaptic release sites

Science

Immunocytochemical localization of candidates for vesicular glutamate transporters in the rat cerebral cortex

J. Comp. Neurol.