Our research efforts are focused on the following main areas:
Glutamate Transporter Physiology and Function
     Neurotransmission at excitatory synapses involves the release of glutamate from vesicles, diffusion and binding of glutamate to receptors, and eventual uptake of glutamate by transporters (see schematic diagram below).
    Transporter-mediated uptake is critical for terminating the actions of glutamate, preventing the sustained activation of receptors that would otherwise disrupt signaling at synapses and lead to excitotoxic neurodegeneration.  At synapses in the CNS, glutamate transporters can influence the occupancy of glutamate receptors by reducing the peak concentration of glutamate within the synaptic cleft, by accelerating the decay of the glutamate transient, and by restricting the diffusion of glutamate to perisynaptic receptors or receptors at adjacent synapses.  The ability of transporters to alter synaptic responses has been shown to depend on the structure of the synapse, the properties of the glutamate receptors, and the frequency of release.  For synapses that contain a high density of release sites, such as calyceal synapses, or are tightly ensheathed by glial processes, transporter inhibition prolongs the decay of EPSCs.  At synapses characterized by partial ensheathment by astrocytes and single release sites, transporter blockers have little effect on the decay of either AMPA or NMDA EPSCs.  These results have lent support to the hypothesis that at some excitatory synapses diffusion, and not uptake, is primarily responsible for rapid dissipation of the glutamate within the synaptic cleft.  The dependence on uptake is often increased when high intensity or high frequency stimuli are applied, or if the probability of release is artificially increased.  Under such circumstances, prolongation of synaptic responses occurs at synapses in the hippocampus and the cerebellum when transporters are inhibited.  These manipulations increase the probability that adjacent synapses release glutamate, and perhaps saturate transporters, resulting in enhanced spillout of transmitter from the synaptic cleft.  The net effect is spillover of transmitter onto receptors at adjacent synapses, and pooling of transmitter between neighboring synapses (crosstalk).  Similarly, receptors that are located further away from release sites, such as perisynaptic or presynaptic metabotropic receptors, are enhanced by transporter inhibition.
     Of the transporters that have been identified, GLAST and GLT-1 are primarily expressed by astroglial cells (astrocytes and Bergmann glial cells), and have similar properties; measurements of Km in heterologous expression systems, or native membranes indicate that these transporters have an affinity for glutamate between 15 and 80 µM.  Astroglial cells are optimized for glutamate uptake, due to their high resting potential and low cytoplasmic concentration of glutamate.  The density of glutamate transporters in hippocampal astrocytes has been estimated to be between 2,500/µm2 and ~10,000/µm2.  The high density of transporters in astrocyte membranes, the high capacity of astrocytes for glutamate uptake, and the close association of astrocyte processes with synapses, suggests that glial transporters are important for removing glutamate released at synapses.  This hypothesis is supported by the activation of these transporters following stimulation of glutamatergic afferents, the dramatic loss in glutamate uptake capacity of brain tissue taken from animals treated with antisense against GLT-1, and the abnormal neurological symptoms of animals that have had the expression of GLT-1 transporters disrupted.  In contrast, transgenic mice lacking EAAC1, the predominant neuronal glutamate transporter, have no CNS phenotype, and the seizure activity observed in adult animals treated with antisense to EAAC1 has been attributed to insufficient GABA synthesis due to a reduction in the uptake of glutamate into GABAergic terminals.  These data support the hypothesis that astrocytes play an essential role in removing glutamate that is released during excitatory transmission.
     There are many unresolved questions about the role of glutamate transporters in synaptic signaling.  Although four glutamate transporters are expressed in the brain, termed EAAT1-4 (EAAT5 is found only in the retina), we do not yet know what the individual contribution of these transporters is to glutamate clearance.  In vitro experiments suggest that glutamate transporters may require tens of milliseconds to complete a cycle, though there have not yet been any measurements of transporter cycling in intact tissue under physiological conditions.  In addition, critical questions remain to be answered regarding the efficiency of transporters (the probability that when a transporter binds a molecule of glutamate, that this molecule is translocated and released into the cytosol), and the speed with which glutamate is removed from the extracellular space.
     Ongoing projects in the lab include studies of the biophysical properties of astroglial transporters (GLT-1 and GLAST), the pathways involved in regulating the activity and expression of glutamate transporters, and the role of specific transporters in excitatory synaptic transmission in the hippocampus, cerebellum, and in the cochlea of the mammalian ear.  We are also developing new methods for monitoring transporter activity using fluorescent imaging.  We rely primarily on whole-cell and patch voltage-clamp recording techniques, which are combined with infrared-DIC imaging to visualize cells in acute brain slices.  Additional approaches involve the rapid application of solutions to outside-out patches using piezoelectric bimorphs, and imaging of intracellular Ca2+ with laser scanning confocal microscopy.
This work is supported by a grant from the NIH/NINDS (NS033958).
Signaling Between Neurons and Glial Cells at Synapses
     The ability of the CNS to integrate sensory input, perform cognitive functions, and coordinate complex motor output is dependent on the rapid transmission of signals over long distances.  This rapid propagation of action potentials is achieved by insulating axons with a sheath of myelin derived from thin membrane sheets of oligodendrocytes.  When myelination is disrupted, transmission along axons is impaired and abnormal neuronal signaling results.  Damage to oligodendrocytes or their progenitors through ischemia, either in utero or following premature birth, can lead to a permanent decrease in motor coordination and impaired cognitive function, termed cerebral palsy.  Recent results indicate that oligodendrocytes and oligodendrocyte progenitors are highly vulnerable to glutamate receptor-mediated excitotoxicity, suggesting a direct role of this neurotransmitter in ischemia-induced injury to myelin.  The susceptibility of infants to white matter damage coincides to a period when oligodendrocytes are at an immature developmental stage.  We recently demonstrated that oligodendrocyte precursor cells (OPCs) form direct glutamatergic synapses with neurons in the hippocampus, suggesting that glutamate receptors are used as a pathway for signaling the state of neural activity to these immature oligodendrocytes.
     Unlike many progenitors in the CNS, OPCs remain numerous in the adult brain long after myelination has concluded, suggesting that they may be retained to replace oligodendrocytes that have been lost through injury or disease.  However, OPCs in the adult brain differ from those present perinatally and show a reduced ability to proliferate and migrate to regions where oligodendrocytes are needed.  Nevertheless, when exposed to serum and growth factors in vitro, purified OPCs can be converted to multipotent stem cells capable of differentiating into both neurons and glia (including oligodendrocytes).  These results raise the exciting possibility that OPCs could be used therapeutically for cell replacement, and highlight the importance of identifying the factors that regulate OPC development in situ.
     Ongoing projects in the lab include characterization of both glutamatergic and gabaergic inputs in these cells during development, analysis of the diversity of OPCs in the CNS, and evaluation of the role of these synaptic inputs in regulating OPC development.  We are also exploring the possibility that OPCs may influence signaling at neuronal synapses.  The techniques employed are simultaneous fluorescence and IR-DIC imaging in brain slices to identify OPCs in different brain areas, whole-cell patch-clamp recordings to record synaptic currents from these cells, and retrospective immunohistochemistry to define the biochemical properties of these cells.
This work is supported by grants from the NIH/NINDS (NS051509) and the Packard Center for ALS Research at Johns Hopkins University.
Glutamate transport in the cochlea
     Auditory signals are transmitted to the brain by converting graded depolarization of inner hair cells (IHCs) to trains of action potentials in auditory nerve fibers. This initial transduction occurs at ribbon synapses between IHCs and afferent dendrites of auditory nerves, in which release of glutamate triggers activation of AMPA receptors. At excitatory synapses in the CNS, glutamate is cleared by high-affinity transporters present in the membranes of neurons and glia. By limiting the accumulation of extracellular glutamate, transporters prevent tonic activation of receptors and excitotoxicity. Transporters also act on a rapid timescale to shape the activation of synaptic and extrasynaptic receptors because they compete with receptors for binding of glutamate as it exits the synaptic cleft. Both immunocytochemical and reverse transcription-PCR analyses indicate that glutamate transporters are expressed in the mammalian organ of Corti; however, the mechanisms of glutamate clearance in the cochlea are not well understood because there have been no functional studies of glutamate transporters in this region.
     In the CNS, glutamate transporters reach their highest density in astrocyte membranes. By extending processes to excitatory synapses, astrocytes create a barrier to diffusion and bring transporters near sites of release. Although most neurons express excitatory amino acid carrier 1 (EAAC1), recent studies indicate that glutamate transporter-1 (GLT-1) and glutamate aspartate transporter (GLAST), two glutamate transporters expressed by astrocytes, are responsible for the majority of glutamate uptake near excitatory synapses. In the mammalian cochlea, astrocyte-like cells, including inner phalangeal cells (IPCs) and border cells located near IHC afferent synapses, exhibit immunoreactivity to GLAST, and GLAST-deficient mice exhibited an impaired recovery from acoustic overstimulation, suggesting that this transporter plays an important role in preventing glutamate accumulation in the cochlea. Nevertheless, the high quantal content of EPSCs at IHC afferent synapses and the high rate of release sustained by IHCs may pose unique challenges for transporter-mediated uptake.
     Ongoing projects in the lab include functional characterization of glutamate transporters in IPCs in the mammalian cochlea, and evaluating the role of the GLAST transporter in shaping EPSCs and high frequency synaptic transmission at the IHC to auditory afferent synapse.
Spontaneous purinergic signaling in the developing cochlea


     Auditory perception depends on the precise conversion of sound-induced vibrations of the basilar membrane into graded release of transmitter from inner hair cells (IHCs), which provide the main excitatory input to auditory nerve fibers. During development, the immaturity of the middle and inner ear prevents the transduction of sound into electrical signals. Nevertheless, auditory nerve fibers periodically fire action potentials before the onset of hearing. The mechanisms responsible for initiating auditory nerve firing in the absence of sound are not well understood.


     We have recently shown that inner hair cells in the developing rat cochlea undergo rhythmic depolarizations, which trigger the release of glutamate and bursts of action potentials in primary auditory afferents. This spontaneous activity is not intrinsic to IHCs, but is rather initiated by the release of ATP from supporting cells within Kölliker’s organ, a columnar epithelium in the inner sulcus that disappears after the onset of hearing. These data indicate that glial-like supporting cells communicate with surrounding sensory cells to initiate electrical activity in auditory afferents, and provide strong evidence that supporting cells play an essential role in the development of auditory pathways. These results also raise the possibility that similar mechanisms might be implicated in response to noise-induced damage and in pathological conditions such as peripheral tinnitus, where sounds are perceived in the absence of auditory stimuli.

     Ongoing projects in the lab include further characterizing spontaneous purinergic signaling in the developing cochlea, and investigating the role of this activity in the development of central auditory pathways.
This work is supported by grants from the NIH/ NIDCD (DC008860 and DC009464) and NINDS (PAR-02-059)