Erik B. Malarkey

Vesicular transmitter release from astrocytes

Astrocytes can release a variety of transmitters, including glutamate and ATP, in response to stimuli that induce increases in intracellular Ca2+ levels. This release occurs via a regulated, exocytotic pathway. As evidence of this, astrocytes express protein components of the vesicular secretory apparatus, including synaptobrevin 2, syntaxin, and SNAP‐23. Additionally, astrocytes possess vesicular organelles, the essential morphological elements required for regulated Ca2+‐dependent transmitter release. The location of specific exocytotic sites on these cells, however, remains to be unequivocally determined.

Ca2+ entry through TRPC1 channels contributes to intracellular Ca2+ dynamics and consequent glutamate release from rat astrocytes

Astrocytes can respond to a variety of stimuli by elevating their cytoplasmic Ca2+ concentration and can in turn release glutamate to signal adjacent neurons. The majority of this Ca2+ is derived from internal stores while a portion also comes from outside of the cell. Astrocytes use Ca2+ entry through store‐operated Ca2+ channels to refill their internal stores. Therefore, we investigated what role this store‐operated Ca2+ entry plays in astrocytic Ca2+ responses and subsequent glutamate release. Astrocytes express canonical transient receptor potential (TRPC) channels that have been implicated in mediating store‐operated Ca2+ entry. Here, we show that astrocytes in culture and freshly isolated astrocytes from visual cortex express TRPC1, TRPC4, and TRPC5. Indirect immunocytochemistry reveals that these proteins are present throughout the cell; the predominant expression of functionally tested TRPC1, however, is on the plasma membrane. Labeling in freshly isolated astrocytes reveals changes in TRPC expression throughout development. Using an antibody against TRPC1 we were able to block the function of TRPC1 channels and determine their involvement in mechanically and agonist‐evoked Ca2+ entry in cultured astrocytes. Blocking TRPC1 was also found to reduce mechanically induced Ca2+‐dependent glutamate release. These data indicate that Ca2+ entry through TRPC1 channels contributes to Ca2+ signaling in astrocytes and the consequent glutamate release from these cells.

Conductive single-walled carbon nanotube substrates modulate neuronal growth.

We used conductive nanotube films as substrates with which we could systematically vary the conductance to see how this property affects neuronal growth. Here we show that nanotube substrates in a narrow range of conductivity promote the outgrowth of neurites with a decrease in the number of growth cones as well as an increase in cell body area, while at higher conductance these effects disappear.

Ca2+-Dependent Glutamate Release Involves Two Classes of Endoplasmic Reticulum Ca2+ Stores in Astrocytes

Astrocytes can modulate synaptic transmission by releasing glutamate in a Ca2+‐dependent manner. Although the internal Ca2+ stores have been implicated as the predominant source of Ca2+ necessary for this glutamate release, the contribution of different classes of these stores is still not well defined. To address this issue, we cultured purified solitary cortical astrocytes and monitored changes in their internal Ca2+ levels and glutamate release into the extracellular space. Ca2+ levels were monitored by using the Ca2+ indicator fluo‐3 and quantitative fluorescence microscopy. Glutamate release was monitored by an L‐glutamate dehydrogenase‐linked detection system. Astrocytes were mechanically stimulated with a glass pipette, which reliably caused an increase in internal Ca2+ levels and glutamate release into the extracellular space. Although we find that the presence of extracellular Cd2+, a Ca2+ channel blocker, significantly reduces mechanically induced glutamate release from astrocytes, we confirm that internal Ca2+ stores are the predominant source of Ca2+ necessary for this glutamate release. To test the involvement of different classes of internal Ca2+ stores, we used a pharmacological approach. We found that diphenylboric acid 2‐aminoethyl ester, a cell‐permeable inositol 1,4,5‐trisphosphate (IP3) receptor antagonist, greatly reduced mechanically induced glutamate release. Additionally, the preincubation of astrocytes with caffeine or ryanodine also reduced glutamate release. Taken together, our data are consistent with dual IP3‐ and caffeine/ryanodine‐sensitive Ca2+ stores functioning in the control of glutamate release from astrocytes.

Applications of Carbon Nanotubes in Neurobiology

Background: Carbon nanotubes are one of the most promising materials for the electronics, computer and aerospace industries. There are numerous properties of carbon nanotubes that make them attractive for applications in neurobiology: small size, flexibility, strength, inertness, electrical conductivity and ease of modification with biological compounds. Objective/Methods: Here, we discuss the current applications of carbon nanotubes in neuroscience. Results: Carbon nanotubes and their derivatives can be used as substrates/scaffolds for neural cell growth. The chemical properties of carbon nanotubes can be systematically varied by attaching different functional groups; manipulation of the charge carried by functionalized carbon nanotubes can be used to control the outgrowth and branching pattern of neuronal processes. The ease with which carbon nanotubes can be patterned makes them attractive for studying the organization of neural networks and the electrical conductivity of nanotubes can provide a mechanism to monitor or stimulate neurons through the substrate itself. However, it is important to recognize that carbon nanotubes themselves can affect neuronal function, most likely by interaction with ion channels. Conclusion: The use of carbon nanotubes in neurobiology is a promising application that has the potential to develop new methods and techniques to advance the study of neuroscience.

Vesicular release of glutamate mediates bidirectional signaling between astrocytes and neurons.

The major excitatory neurotransmitter in the CNS, glutamate, can be released exocytotically by neurons and astrocytes. Glutamate released from neurons can affect adjacent astrocytes by changing their intracellular Ca2+ dynamics and, vice versa, glutamate released from astrocytes can cause a variety of responses in neurons such as: an elevation of [Ca2+]i, a slow inward current, an increase of excitability, modulation of synaptic transmission, synchronization of synaptic events, or some combination of these. This astrocyte‐neuron signaling pathway might be a widespread phenomenon throughout the brain with astrocytes possessing the means to be active participants in many functions of the CNS. Thus, it appears that the vesicular release of glutamate can serve as a common denominator for two of the major cellular components of the CNS, astrocytes and neurons, in brain function.

Water Soluble Single-Walled Carbon Nanotubes Inhibit Stimulated Endocytosis in Neurons

We report the use of chemically functionalized water soluble single-walled carbon nanotube (SWNT) graft copolymers to inhibit endocytosis. The graft copolymers were prepared by the functionalization of SWNTs with polyethylene glycol. When added to the culturing medium, these functionalized water soluble SWNTs were able to increase the length of various neuronal processes, neurites, as previously reported. Here we have determined that SWNTs are able to block stimulated membrane endocytosis in neurons, which could then explain the previously noted extended neurite length.

Leptin resistance is a secondary consequence of the obesity in ciliopathy mutant mice

Although primary cilia are well established as important sensory and signaling structures, their function in most tissues remains unknown. Obesity is a feature associated with some syndromes of cilia dysfunction, such as Bardet-Biedl syndrome (BBS) and Alström syndrome, as well as in several cilia mutant mouse models. Recent data indicate that obesity in BBS mutant mice is due to defects in leptin receptor trafficking and leptin resistance. Furthermore, induction of cilia loss in leptin-responsive proopiomelanocortin neurons results in obesity, implicating cilia on hypothalamic neurons in regulating feeding behavior. Here, we directly test the importance of the cilium as a mediator of the leptin response. In contrast to the current dogma, a longitudinal study of conditional Ift88 cilia mutant mice under different states of adiposity indicates that leptin resistance is present only when mutants are obese. Our studies show that caloric restriction leads to an altered anticipatory feeding behavior that temporarily abrogates the anorectic actions of leptin despite normalized circulating leptin levels. Interestingly, preobese Bbs4 mutant mice responded to the anorectic effects of leptin and did not display other phenotypes associated with defective leptin signaling. Furthermore, thermoregulation and activity measurements in cilia mutant mice are inconsistent with phenotypes previously observed in leptin deficient ob/ob mice. Collectively, these data indicate that cilia are not directly involved in leptin responses and that a defect in the leptin signaling axis is not the initiating event leading to hyperphagia and obesity associated with cilia dysfunction.

Temporal characteristics of vesicular fusion in astrocytes: examination of synaptobrevin 2‐laden vesicles at single vesicle resolution

Non‐technical summary  Astrocytes have been shown to release transmitters by vesicle fusion, in a manner similar to that of neuronal exocytosis. The details of this process in astrocytes are not well understood, so we used a fluorescently labelled vesicle protein, synapto‐pHluorin (spH), to track how these fusions occurred. When astrocytes were mechanically stimulated we saw a slow burst of fusions, while other stimuli caused a relatively even sustained rate of fusion. We observed two distinct types of events, transient and full fusions, the proportion of which was stimulus dependent. Similarly, stability of the vesicle fusion pore with the plasma membrane varied with the stimulus. We describe the effects on fusion events resulting from expressing variants of exocytotic proteins, synaptotagmin 1 and SNAP25B. Studying the characteristics of astrocytic exocytosis will aid in the general understanding of this process and also events at the tripartite synapse, both in health and disease.

Carbon nanotubes in neuroscience.

Carbon nanotubes have electrical, mechanical and chemical properties that make them one of the most promising materials for applications in neuroscience. Single-walled and multi-walled carbon nanotubes have been increasingly used as scaffolds for neuronal growth and more recently for neural stem cell growth and differentiation. They are also used in interfaces with neurons, where they can detect neuronal electrical activity and also deliver electrical stimulation to these cells. The emerging picture is that carbon nanotubes do not have obvious adverse effects on mammalian health. Thus in the near future they could be used in brain-machine interfaces.