Material below is adapted from the SfN Short Course Advances in Multi-Neuronal Monitoring of Brain Activity, by Douglas S. Kim, PhD, Vivek Jayaraman, PhD, Loren L. Looger, PhD, and Karel Svoboda, PhD. Short Courses are daylong scientific trainings on emerging neuroscience topics and research techniques held the day before SfN’s annual meeting.

As neuroscientists try to understand the dynamics of both large networks of neurons and single cells, new methods to monitor neuronal activity are being developed. One way to study synaptic and action potential activity is to measure intracellular calcium. Within dendritic spines, calcium transients correlate with synaptic activity, and in cell bodies, calcium signals accurately reflect action potentials (spikes). 

Early techniques to visualize calcium involved synthetic dyes that emit light in proportion to the amount of calcium present. But these dyes are not specific in that astrocytes and other cells are also labeled. In addition, synthetic dyes cannot be used for repeated, long-term imaging. Newer sensors such as genetically encoded calcium indicators (or GECIs) are delivered by viruses or bred into transgenic animals, allowing expression that can be both long term and targeted to specific cell types. They can be used both in cell cultures and living animals and optimized for specific research questions. 

One such GECI is GCaMP, a fusion protein linking green fluorescent protein to calmodulin and the M13 domain of myosin light chain kinase. When calcium binds calmodulin, calmodulin binds M13. These interactions change the structure of the protein complex, allowing it to glow green where the brightness is proportional to the increase in calcium. 

At first, GECIs had poor temporal resolution, but sensitivity has improved dramatically. For example, when mice were shown different stimuli, one GCaMP variant was able to detect single spikes in visual cortical neurons. Furthermore, the same neurons could be imaged for months. 

To optimize imaging for different cell types, future manipulations will also tweak binding affinity. In cells with high frequency activity, the signals get easily saturated; a lower binding affinity would improve detection. Alternatively, a higher affinity would be helpful in cells in which calcium is quickly buffered, like parvalbumin-expressing interneurons.

In addition to green fluorescent calcium sensors, red fluorescent sensors have also been created. Red light can penetrate farther into tissue, enabling deeper and less invasive imaging than green. Red is also more compatible with common optogenetic methods that use blue light, like channelrhodopsin-2-based experiments. Newer red GECIs are beginning to approach the sensitivity of green GECIs, although bleaching remains a problem. 

As GECIs continue to improve, future possibilities for the technology are exciting. For example, combining red and green sensors will allow imaging of multiple cell types simultaneously. GECIs provide researchers a way to witness the dynamics of cell communication across networks and within sub-compartments of an individual cell in a whole new light.

Material below summarizes the article Dopamine Modulates the Activity of Sensory Hair Cells, published on December 16, 2015, in JNeurosci and authored by Cecilia Toro, Josef G. Trapani, Itallia Pacentine, Reo Maeda, Lavinia Sheets, Weike Mo, and Teresa Nicolson.

Perception of sensory stimuli, like sounds waves, is typically thought of as a unidirectional process: Sound waves hits the eardrum, and are then sensed by the inner ear, which conveys those auditory signals to the brain. At various centers of the brain, these signals are processed and relevant information is sorted. This allows for focused perception, such as hearing a particular voice among many voices.

However, there is just as much, if not more, communication that happens in the opposite direction. The brain also signals back to the ear and can influence its activity. For example, if a person is exposed to continuous loud noise, then the auditory system adapts. Consider how loud a rock concert would seem if you walked into the concert hall from a quiet room versus how loud you would perceive it to be 10 minutes later. This adaptation occurs in part by signaling from the brain to the ear.

Communication and feedback from the brain to the ear is, in most cases, not very well understood. Generally, the same signaling molecules that mediate communication among neurons in the brain are also used for signaling to the ear.

One such molecule is the neurotransmitter dopamine, which is better known for its roles in the brain’s reward system and Parkinson’s disease. Dopamine is present in the ear, but how this neurotransmitter affects cellular function and signaling has not been fully explored, partially because of the complexities of the inner ear. The inner ear expresses multiple types of dopamine receptors that signal in different ways upon binding of dopamine.

To get around this issue, we took advantage of the relatively simple system of the lateral line organ in zebrafish larvae, which is closely related to the inner ear in terms of function and cellular mechanisms.

The inner ear and lateral line are both mechanosensory organs — they respond to specific physical stimuli. 

The inner ear stimulus is the movement of fluid within the cochlea or the semi-circular canals that is caused by sound waves hitting the eardrum or head movement.

The lateral line, on the other hand, is used by most aquatic vertebrates to detect the movement of external water. This is essential for vital zebrafish behaviors like schooling, predator and prey detection, and body orientation in currents. The mechanosensory cells (hair cells) of the lateral line organ are analogous to those that are employed by the ear to sense sounds and head movements. Therefore, the zebrafish lateral line organ is a good model system to study the cells and molecules that govern the senses of hearing and balance.

We discovered that only one type of dopamine receptor is present in the lateral line organ — the D1 receptor. We used a number of methods, including recording the charge movement through hair cells (using electrophysiology) and the concentration of calcium ions (using a sensitive imaging technique that detects fluorescence resonance energy transfer ‘FRET imaging’), to try to discern what the function of the D1 dopamine receptor is in the lateral line organ.

We observed that dopamine enhances the activity of the mechanosensory hair cells. The same mechanical stimulation produces a greater response in hair cells if dopamine receptors are activated. We know that the concentration of calcium ions is intimately linked to the ability of hair cells to send sensory signals to the brain. Therefore, we asked whether dopamine was working by increasing the movement of calcium ions into hair cells. Our calcium imaging experiments suggest that enhancement of activity depends on calcium channels that mediate calcium influx into hair cells. Finally, we looked at the shape and location of the axons that likely release dopamine in the lateral line to better understand how they might communicate with hair cells.

Our evidence suggests that a single neuron may communicate with numerous hair cells at once by releasing dopamine into the tissue surrounding multiple cells. This type of release supports the idea of dopamine exerting global control over the lateral line system, rather than fine-tuning any one hair cell’s activity. We also found that hair cells of the zebrafish inner ear express the same type of D1 dopamine receptor, suggesting that a similar mechanism is working in auditory and vestibular hair cells.

Why would enhancement of sensitivity to sound or head movements by dopamine be a useful mechanism?

The answer to this question is unknown, but the global nature of the dopamine signaling in the lateral line organ, along with the origination of these dopaminergic neurons in a particular part of the brain, suggests that this feedback system might have to do with ensuring the function or maintenance of the system (homeostasis), or that sensitivity may be affected by circadian rhythms. Perhaps having increased hearing sensitivity during the day would be an advantage for diurnal animals. Exploration of the biological function of dopamine signaling in the ear awaits further investigation.

Visit JNeurosci to read the original article and explore other content. Read other summaries of JNeurosci and eNeuro papers in the Neuronline collection SfN Journals: Research Article Summaries.

Dopamine Modulates the Activity of Sensory Hair Cells. Cecilia Toro, Josef G. Trapani, Itallia Pacentine, Reo Maeda, Lavinia Sheets, Weike Mo, Teresa Nicolson. The Journal of Neuroscience Dec 2015, 35(50): 16494-16503; DOI: 10.1523/JNEUROSCI.1691-15.2015

Material below summarizes the article Simultaneous Sodium and Calcium Imaging from Dendrites and Axons, published on October 14, 2015, in eNeuro and authored by Kenichi Miyazaki and William N. Ross.

Dynamic calcium imaging is an important technique. It can reveal information about the location of various calcium channels and calcium permeable receptors; the time course, magnitude, and location of intracellular calcium concentration ([Ca²⁺]_i) changes; and indirectly, the occurrence of action potentials.

Dynamic sodium imaging, a less-used technique, can reveal analogous information related to sodium signaling. In some cases, like the imaging of AMPA and NMDA receptor responses to synaptic stimulation, detection of both [Ca²⁺]_i and [Na⁺]_i changes in the same preparation may provide more information than separate measurements. This is because both receptors are permeable to sodium, but only the NMDA receptor is permeable to calcium. 

Previously, investigators interested in measuring both kinds of ion changes would do separate experiments with the hope that the conditions in both would be the same: one measuring [Ca²⁺]_i changes and, in a separate preparation, an experiment measuring [Na⁺]_i changes. Since this hope could not be guaranteed, the experiments would have to be interpreted carefully. If both measurements could be made at the same time, then more precise comparisons could be made. 

We developed a technique to simultaneously measure both signals at high speed and sufficient sensitivity to detect localized physiological events. We first looked for fluorescent indicator molecules for [Ca²⁺]_i and [Na⁺]_i changes that have separated excitation bands. Two combinations met this requirement: SBFI, a sodium sensitive indicator excited near 365 nm and any of the Oregon Green or Fluo calcium indicators excited near 480 nanometers; or any of the fura series of indicators excited near 380 nanometers and ANG-2, a sodium indicator excited near 520 nanometers. By switching between the two excitation wavelengths, we could excite one indicator or the other if they were both loaded into the same cell. 

We also designed custom dichroic and emission filters to allow the optimal separate detection of the fluorescence of sodium and calcium indicators. To excite the two indicators quasi-simultaneously, we alternated the wavelength of excitation rapidly (500 hertz). We achieved this high speed using high power light emitting diodes (LEDs). These LEDs turn on and off in microseconds, have relatively narrow emission bands, and emit intensities much higher than arc lamps at the important wavelengths. To detect the alternating flashes of fluorescence from the two indicators, we synchronized the two millisecond LED pulses with movie frames from a sensitive, low noise, high speed CCD camera (RedShirtImaging NeuroCCD-SMQ) operated at 500 hertz. 

Software then separated the two data streams from alternating frames to provide independent sodium and calcium signals. Additional channels recorded the voltage and current traces from the patch electrode that was also used to inject the indicators into the cell. The analysis program then displayed all four channels (two optical and two electrical) on the same time base.

This system can be used to detect simultaneous signals from any two fluorescent indicators that have separated excitation bands. In the paper, we give examples where we measured [Ca²⁺]_i changes with two different calcium indicators and [Na⁺]_i with different sodium indicators. These kinds of measurements might be useful if, for example, one indicator was high affinity (a good choice for measuring small concentration changes) and the other indicator was low affinity (a good choice for non-saturating and linear measurements). In principle, this system could be extended to record three optical channels simultaneously, provided the excitation bands are sufficiently separated. A practical example would be detection of [Ca²⁺]_i changes with high and low affinity indicators together with the detection of [Na⁺]_i changes. Finally, it should be noted that ours is not the only system capable of simultaneously detecting signals from two sources. Other systems, using different approaches, are referenced in the paper. 

Our current experiments use this system to examine dendritic properties of pyramidal neurons in rat hippocampal slices. In a typical experiment we can detect [Ca²⁺]_i and [Na⁺]_i changes from single action potentials in axons and synaptically evoked signals in dendrites, both with submicron resolution and a good signal to noise ratio. We often find that the two signals give complementary information about the cell response. 

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Simultaneous Sodium and Calcium Imaging from Dendrites and Axons. Kenichi Miyazaki, William N. Ross. eNeuro Oct 2015, 2 (5). DOI: 10.1523/ENEURO.0092-15.2015

About the Contributors

William Ross, PhD

William Ross is a professor in the physiology department at New York Medical College.

Kenichi Miyazaki, PhD

Kenichi Miyazaki is a postdoctoral fellow at New York Medical College.

Material below summarizes the article NEUROD2 Regulates Stim1 Expression and Store-Operated Calcium Entry in Cortical Neurons, published on February 27, 2017, in eNeuro and authored by Gokhan Guner, Gizem Guzelsoy, Fatma Sadife Isleyen, Gulcan Semra Sahin, Cansu Akkaya, Efil Bayam, Eser Ilgin Kotan, Alkan Kabakcioglu, and Gulayse Ince-Dunn.

The cerebral cortex is the seat of higher cognitive functions such as learning, memory, planning, language acquisition, attention, and consciousness. In order to carry out these complex tasks, the cortex is composed of myriad types of neurons amounting to approximately 20-25 billion in humans.

A large proportion of these neurons — excitatory neurons — use the excitatory neurotransmitter glutamate and assemble into circuits that function in local communications and long-distance communications with other parts of the brain. A formidable task for the developing cerebral cortex is the production, differentiation, and assembly of excitatory cortical neurons into properly functioning networks.

A unique feature of brain development is that brain development is not only shaped by inherited genetic instructions but also by neuronal activity that is either spontaneous or driven by sensory experience. Calcium-regulated transcription factors emerge as integrators of the cellular effects of neuronal activity and transduce them into changes in gene expression programs.

Our interest in such a factor began when NEUROD2 was identified by a screen designed to clone calcium-regulated transcriptional activators in cortical neurons. NEUROD2 was then characterized as a transcription factor that is highly expressed in the differentiating excitatory neurons of the cortex.

Since its original cloning, a number of studies demonstrated NEUROD2’s essential role in various aspects of cortical development, such as the formation of neural maps that represent sensory stimuli and the maturation of dendrites and synapses. We reasoned that a potentially informative approach to understanding the cellular effects of neuronal activity-regulated gene expression would be to identify the genome-wide targets of a transcription factor such as NEUROD2.  

To achieve this goal, we applied a ChIP-Seq (chromatin immunoprecipitation and sequencing) approach to identify genomic locations that were directly bound by NEUROD2 within the cerebral cortex of newly born mice pups. ChIP-Seq is a biochemical purification method that relies on immunoprecipitation of covalently crosslinked DNA binding proteins of interest to their target sequences followed by high-throughput sequencing of associated DNA.

As expected, our computational analyses of all NEUROD2 binding sites revealed that a large proportion of them mapped onto promoter regions of target genes. Unexpectedly, however, we also observed that even a larger proportion of binding events were localized to non-promoter regions located within genes or at intergenic regions.

We reasoned that at least a subset of these binding events might correspond to cis-regulatory elements. These are DNA sequences that are typically non-coding and bind multiple transcription factors to regulate gene expression in a manner that is specific to a particular cell type, developmental stage, or external stimulus. Therefore, we focused our attention to a top NEUROD2 binding site that was localized to a potential cis-regulatory element.

This top NEUROD2 binding site was localized within an intron of a gene called Stim1 (Stromal Interaction Molecule 1). Interestingly, the protein product of Stim1 gene encodes a regulator of a particular pathway of calcium entry into neurons. This pathway, named store-operated calcium entry (SOCE), is composed of calcium influx through plasma membrane calcium channels which are specifically activated by the emptying of endoplasmic reticulum calcium stores. STIM1 protein is a sensor of ER calcium content, and upon ER emptying, it induces calcium influx from the external milieu.

Initially, we questioned how Stim1 expression was impacted by NEUROD2. Upon silencing of Neurod2 in primary cortical neurons, we observed a significant increase in Stim1 expression at the mRNA and protein level, suggesting that NEUROD2 acts as a suppressor of Stim1 gene.

Our results suggested a testable physiological hypothesis, which is that NEUROD2 fine-tunes neuronal SOCE. To address this intriguing possibility, we carried out live calcium-imaging experiments following knockdown or overexpression of NEUROD2.

Indeed, our results demonstrated that while suppression of NEUROD2 expression upregulates SOCE, its overexpression augments SOCE. Thus, our results demonstrated that NEUROD2 is a critical regulator of neuronal SOCE.

Store-operated calcium entry is attracting increased interest from the neuroscience community in light of recent studies demonstrating its role in guiding the growth of axons, migration of neurons, and the pathogenesis of certain neurological diseases like epilepsy and Alzheimer’s disease.

In contrast to other sources of calcium influx, very little is known about the mechanisms by which SOCE regulates signaling and many aspects of physiological and morphological maturation of neurons. Specifically, how SOCE itself is regulated by upstream gene expression programs in neurons is essentially completely unknown.

We hypothesize that a feedback regulatory loop might exist in neurons for the regulation of SOCE. In such a scenario, calcium influx activates NEUROD2, which in turn suppresses Stim1 expression, thereby inhibiting an essential component of store-operated calcium entry.

Our future experiments will focus on directly testing this intriguing possibility.

Visit eNeuro to read the original article and explore other content. Read other summaries of JNeurosci and eNeuro papers in the Neuronline collection SfN Journals: Research Article Summaries.

NEUROD2 Regulates Stim1 Expression and Store-Operated Calcium Entry in Cortical Neurons. Gokhan Guner, Gizem Guzelsoy, Fatma Sadife Isleyen, Gulcan Semra Sahin, Cansu Akkaya, Efil Bayam, Eser Ilgin Kotan, Alkan Kabakcioglu, Gulayse Ince-Dunn. eNeuro Feb 2017, 4 (1) DOI: 10.1523/ENEURO.0255-16.2017

About the Contributor

Gulayse Ince-Dunn

Gulayse Ince-Dunn is an assistant professor at Koç University in Istanbul, Turkey.

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