We are interested in processes that affect how synapses in the central nervous system operate. Our aims are to address fundamental questions such as how neurotransmitters are packaged in to vesicles, what regulates their release, how they interact with the postsynaptic targets and surrounding glial cells, and how they are recycled or otherwise replenished in the presynaptic terminal.
Our lab investigates these questions using a combination of research techniques, including electrophysiological recordings, fluorescent ion imaging and immunohistochemistry. The majority of work is conducted on living cells in acutely isolated brain stem slices. This brain region contains the calyx of Held, a giant synapse in the auditory system. The calyx of Held presynaptic terminal is large enough to be recorded from using whole-cell patch-clamp methods as well as introducing fluorescent molecules to record ion concentration changes and visualise it using wide field or confocal microscopy. In addition to recording from the presynaptic terminal, we also record the electrial activity and ion concentration changes of the postsynaptic MNTB neuron and the surrounding glial cells (astrocytes).
- Figure 1: The calyx of Held synapse in the auditory brainstem circuit.
a Representation in the coronal plane of the brainstem auditory pathway and the calyx of Held synapse, which forms part of the auditory circuit at the level of the superior olivary complex (SOC). The calyx of Held is an excitatory glutamatergic synapse arising from globular bushy cells in the anterior ventral cochlear nucleus (aVCN) onto a principal cell in the medial nucleus of the trapezoid body (MNTB). The principal cells provide an inhibitory projection to other nuclei of the SOC such as the lateral superior olive (LSO). The bushy cells in the aVCN receive excitatory input from the auditory nerve fibres. The calyx of Held is thus a tertiary auditory synapse that rapidly relays information, providing the LSO and other nuclei with (inhibitory) information with regard to sound arriving at the contralateral ear.
b Representation of a single calyx of Held synapse onto a given single MNTB principal cell (modified, with permission, from Elsevier, from Walmsley et al. 1998). The MNTB principal cells receive additional inhibitory and excitatory input through small bouton-like synapses but, in most cases, a given MNTB principal cell is thought to receive input from only one large calyx of Held. Thus, a one-to-one synaptic relationship exists between a given globular bushy cell and an MNTB principal cell. From Schneggenburger and Forsythe (2006) Cell Tissue Res 326:311–337
The calyx of Held presynaptic terminal is one of the largest terminals in the central nervous system. It releases the excitatory neurotransmitter glutamate and can reliably sustain communication at over 500Hz. Neurotransmitter release is triggered by Ca2+ influx through voltage-gated channels. Factors regulating the spatiotemporal profile of the presynaptic cytoplasmic calcium transient influence release probability, vesicle recycling and information processing in the brain. Mitochondria are capable of having an important effect on these processes because they can sequester large quantities of calcium, are present at high concentrations in presynaptic terminals, and can be tethered to vesicle release sites. Our work demonstrates the importance of mitochondria in presynaptic terminals for regulating synaptic release. Our results suggest a new role for presynaptic mitochondria in maintaining transmission by accelerating recovery from synaptic depression after periods of moderate activity. Without detectable thapsigargin-sensitive presynaptic calcium stores, we conclude that mitochondria are the major organelle regulating presynaptic calcium at central glutamatergic terminals.
- Figure 2: Mitochondrial inhibition affects neurotransmitter release. A) DIC image of an MNTB neuron and calyx of Held presynaptic terminal, both with attached patch pipette. B) Fluorescent image of rhodamine and lucifer yellow in the post and pre synaptic cells. C) Presynaptic stimulation (1ms depolarization of presynaptic terminal, 20 times at 200 Hz) results in a postsynaptic EPSC (red). The amount of synaptic depression during the train is increased and the rate of recovery following the train is slowed by inhibiting mitochondrial calcium transport (black). Taken from Billups and Forsythe 2002
The calyx of Held presynaptic terminal releases the excitatory neurotransmitter glutamate. Vesicular glutamate transporters (VGLUTs) are responsible for the accumulation of glutamate into synaptic vesicles. Immunohistochemical staining has been used in the lab to show the developmental changes in expression of the vesicular glutamate transporters VGLUT1 and VGLUT2 in the auditory brainstem. We show that throughout its postnatal developmental period a single presynaptic calyx of Held terminal 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.
- Figure 3: VGLUT expression in the SOC. VGLUT1 (green) and VGLUT2 (red) immunoreactivity in the auditory brainstem of a 29 day old rat. a) The ventral edge of the brainstem from the midline (left edge) to the lateral edge of the slice. b-e) Images of the MNTB, MSO, LSO and VNTB shown at the same higher magnification. f) Flattened stack of 21 confocal sections, 0.5mm apart, through a calyx of Held. From Billups (2005)
The mechanisms by which the excitatory neurotransmitter glutamate is recycled at synapses are currently unknown and are a main focus of investigation in our lab. In line with other studies, we show that glutamate is not directly taken up back into the synaptic terminal by electrogenic glutamate transporters. Instead, radiotracing studies suggest that released glutamate is sequestered by astrocytes, which is then converted into glutamine and transported back to the presynaptic terminal, forming the glutamate-glutamine cycle. However, the nature of the transporters that mediate the translocation of these amino acids has not been established. Our lab shows that astrocytes adjacent to the synapse contains functional high affinity glutamate transporters on their plasma membrane, allowing them to sequester glutamate released during synaptic activity. Moreover, we discovered that these astrocytes can release glutamine in a temporally precise, controlled manner in response to glial glutamate transporter activation, representing a potential feedback mechanism by which astrocytes can respond to synaptic activation and react in a way that sustains or enhances further communication. Furthermore, we show that the glutamine released from the astrocyte is taken up by the neighbouring postsynaptic MNTB neurone via the sodium-dependent electrogenic amino acid transporter system-A, providing these cells with a substrate for amino acid and neurotransmitter metabolism. This neuronal glutamine uptake happens rapidly in response to astrocytic glutamine release, demonstrating a robust signalling mechanism between glia and neurons. Our results unveil a novel feedback mechanism by which astrocytes can potentially modulate neuronal function, and pave the way for development of new therapeutic approaches to treat neurological disorders (Uwechue et al. 2012).
Figure 4: A proposed mechanism for astrocytic glutamine release. Activation of glial glutamate transporters (EAATs) causes a rise in glial [Na+]i, which favours the efflux of glutamine via glial system N transporters (SN). Released glutamine is subsequently detected by system A transporters (SA) located on adjacent MNTB neurones, resulting in a neuronal glutamine transport current (from Uwechue et al. 2012).
Current ongoing research aims include A) elucidating the mechanism of glutamine release from perisynaptic astrocytes and understanding its regulation; and B) determining how presynaptic terminals sequester glutamine and understanding the role this plays is producing glutamate for sustaining excitatory neurotransmission.