Vesicular Glutamate Transporter 2 Is Required for Central Respiratory Rhythm Generation But Not for Locomotor Central Pattern Generation

Department of Neuroscience, Unit of Developmental Genetics, Uppsala University, 751 23 Uppsala, Sweden.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.34). 12/2006; 26(47):12294-307. DOI: 10.1523/JNEUROSCI.3855-06.2006
Source: PubMed


Glutamatergic excitatory neurotransmission is dependent on glutamate release from presynaptic vesicles loaded by three members of the solute carrier family, Slc17a6-8, which function as vesicular glutamate transporters (VGLUTs). Here, we show that VGLUT2 (Slc17a6) is required for life ex utero. Vglut2 null mutant mice die immediately after birth because of the absence of respiratory behavior. Investigations at embryonic stages revealed that neural circuits in the location of the pre-Bötzinger (PBC) inspiratory rhythm generator failed to become active. However, neurons with bursting pacemaker properties and anatomical integrity of the PBC area were preserved. Vesicles at asymmetric synapses were fewer and malformed in the Vglut2 null mutant hindbrain, probably causing the complete disruption of AMPA/kainate receptor-mediated synaptic activity in mutant PBC cells. The functional deficit results from an inability of PBC neurons to achieve synchronous activation. In contrast to respiratory rhythm generation, the locomotor central pattern generator of Vglut2 null mutant mice displayed normal rhythmic and coordinated activity, suggesting differences in their operating principles. Hence, the present study identifies VGLUT2-mediated signaling as an obligatory component of the developing respiratory rhythm generator.

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    • "The preB€ otzinger Complex (preB€ otC) has been identified as a region necessary and sufficient for the endogenous rhythmic respiratoryrelated output generated in isolated neonatal rodent hindbrain preparations (Smith et al., 1991). Synaptic glutamate release from vesicular glutamate transporter 2 (VGlut2)-expressing neurons within the preB€ otC is necessary for the expression of respiratory behaviors both in vivo and in vitro (Greer et al., 1991; Wallen-Mackenzie et al., 2006, 2010). In addition to glutamate, hindbrain neurons release other neurotransmitters including GABA and serotonin, and neuromodulators such as somatostatin (SST) and substance P (SP). "
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    ABSTRACT: Identifying neurons essential for the generation of breathing and related behaviors such as vocalisation is an important question for human health. The targeted loss of preBötzinger Complex (preBötC) glutamatergic neurons, including those that express high levels of somatostatin protein (SST neurons), eliminates normal breathing in adult rats. Whether preBötC SST neurons represent a functionally specialised population is unknown. We tested the effects on respiratory and vocal behaviors of eliminating SST neuron glutamate release by Cre-Lox-mediated genetic ablation of the vesicular glutamate transporter 2 (VGlut2). We found the targeted loss of VGlut2 in SST neurons had no effect on viability in vivo, or on respiratory period or responses to neurokinin 1 or μ-opioid receptor agonists in vitro. We then compared medullary SST peptide expression in mice with that of two species that share extreme respiratory environments but produce either high or low frequency vocalisations. In the Mexican free-tailed bat, SST peptide-expressing neurons extended beyond the preBötC to the caudal pole of the VII motor nucleus. In the naked mole-rat, however, SST-positive neurons were absent from the ventrolateral medulla. We then analysed isolation vocalisations from SST-Cre;VGlut2(F/F) mice and found a significant prolongation of the pauses between syllables during vocalisation but no change in vocalisation number. These data suggest that glutamate release from preBötC SST neurons is not essential for breathing but play a species- and behavior-dependent role in modulating respiratory networks. They further suggest that the neural network generating respiration is capable of extensive plasticity given sufficient time.
    Full-text · Article · Jul 2014 · European Journal of Neuroscience
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    • "The current study showing a BDNF-induced upregulation of total VGLUT protein levels in hippocampal neurons, in addition to an increase in the punctate distribution of the transporters along neurites, provides further evidence indicating a role of this neurotrophin on presynaptic potentiation of glutamatergic transmission. The following evidences suggest that BDNF-induced upregulation in VGLUT clustering in neurites may significantly potentiate excitatory neurotransmission: 1) VGLUT expression directly correlates with synaptic strength [43], [44] and biogenesis or recycling of synaptic vesicles [42], [98]; 2) VGLUT1 deficient mice exhibit decreased spontaneous glutamate release and quantal synaptic transmission due to exocytosis of partially filled vesicles in hippocampal synapses [43]; 3) VGLUT1 overexpression not only rescues this phenotype but also enhances AMPA receptor-mediated evoked EPSCs by increasing glutamate release per vesicle [44]; 4) loss of VGLUT 1 and 2 causes changes in synaptic vesicle shape and leads to decreased number of vesicles [42], [98]; 5) VGLUT2 deficiency decreases evoked glutamate release probability and reduces LTD at hippocampal CA3-CA1 synapses of young postnatal (P11–P14) mice [99]; 6) even though one transporter apparently suffices to fill a vesicle [100], enhanced VGLUT expression may increase the number of transporters per vesicle, thus, accelerating the rate of vesicle filling or its volume [101]. Conversely, decreased VGLUT1 expression causes depressive behavior and impaired memory in mice [47], while VGLUT2 heterozygotes show decreased neuropathic pain and defense responses [98], [102]. "
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    ABSTRACT: BDNF is a pro-survival protein involved in neuronal development and synaptic plasticity. BDNF strengthens excitatory synapses and contributes to LTP, presynaptically, through enhancement of glutamate release, and postsynaptically, via phosphorylation of neurotransmitter receptors, modulation of receptor traffic and activation of the translation machinery. We examined whether BDNF upregulated vesicular glutamate receptor (VGLUT) 1 and 2 expression, which would partly account for the increased glutamate release in LTP. Cultured rat hippocampal neurons were incubated with 100 ng/ml BDNF, for different periods of time, and VGLUT gene and protein expression were assessed by real-time PCR and immunoblotting, respectively. At DIV7, exogenous application of BDNF rapidly increased VGLUT2 mRNA and protein levels, in a dose-dependent manner. VGLUT1 expression also increased but only transiently. However, at DIV14, BDNF stably increased VGLUT1 expression, whilst VGLUT2 levels remained low. Transcription inhibition with actinomycin-D or α-amanitine, and translation inhibition with emetine or anisomycin, fully blocked BDNF-induced VGLUT upregulation. Fluorescence microscopy imaging showed that BDNF stimulation upregulates the number, integrated density and intensity of VGLUT1 and VGLUT2 puncta in neurites of cultured hippocampal neurons (DIV7), indicating that the neurotrophin also affects the subcellular distribution of the transporter in developing neurons. Increased VGLUT1 somatic signals were also found 3 h after stimulation with BDNF, further suggesting an increased de novo transcription and translation. BDNF regulation of VGLUT expression was specifically mediated by BDNF, as no effect was found upon application of IGF-1 or bFGF, which activate other receptor tyrosine kinases. Moreover, inhibition of TrkB receptors with K252a and PLCγ signaling with U-73122 precluded BDNF-induced VGLUT upregulation. Hippocampal neurons express both isoforms during embryonic and neonatal development in contrast to adult tissue expressing only VGLUT1. These results suggest that BDNF regulates VGLUT expression during development and its effect on VGLUT1 may contribute to enhance glutamate release in LTP.
    Full-text · Article · Jan 2013 · PLoS ONE
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    • "Mice with a loss-of-function mutation in the NMDA receptor NR 1 are able to generate normal respiratory rhythm (Funk et al., 1997) but show depressed breathing at birth (Poon et al., 2000). Mice lacking all vGLUT2-dependent glutamatergic transmission fail to produce a respiratory rhythm (Wallén-Mackenzie et al., 2006). Neurons in the pFRG/RTN express vglut2, and developmental defects in the pFRG/RTN observed in Phox2b mutant and Math1 null mice depress breathing (Dubreuil et al., 2008; Rose et al., 2009a). "
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    ABSTRACT: Neural networks called central pattern generators (CPGs) generate repetitive motor behaviors such as locomotion and breathing. Glutamatergic neurons are required for the generation and inhibitory neurons for the patterning of the motor activity associated with repetitive motor behaviors. In the mouse, glutamatergic V2a neurons coordinate the activity of left and right leg CPGs in the spinal cord enabling mice to generate an alternating gait. Here, we investigate the role of V2a neurons in the neural control of breathing, an essential repetitive motor behavior. We find that, following the ablation of V2a neurons, newborn mice breathe at a lower frequency. Recordings of respiratory activity in brainstem-spinal cord and respiratory slice preparations demonstrate that mice lacking V2a neurons are deficient in central respiratory rhythm generation. The absence of V2a neurons in the respiratory slice preparation can be compensated for by bath application of neurochemicals known to accelerate the breathing rhythm. In this slice preparation, V2a neurons exhibit a tonic firing pattern. The existence of direct connections between V2a neurons in the medial reticular formation and neurons of the pre-Bötzinger complex indicates that V2a neurons play a direct role in the function of the respiratory CPG in newborn mice. Thus, neurons of the embryonic V2a lineage appear to have been recruited to neural networks that control breathing and locomotion, two prominent CPG-driven, repetitive motor behaviors.
    Full-text · Article · Jun 2012 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
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