SFN: Neuroethics: Hank Greely on the impact of neuroscience on society

First up for me this morning: Hank Greely's talk entitled The Neuroscience Revolution and Society. For those of you not familiar with Hank Greely, he is a law professor at Stanford University who is (to quote his faculty website), a "leading expert on the legal, ethical, and social issues surrounding health law and the biosciences" who specializes in  the"implications of new biomedical technologies, especially those related to neuroscience, genetics, and stem cell research." I have heard Greely speak 2 times previously, discussing the implications of neuroscience (in particular fMRI technology) for society and the law. Last month, Greely chaired a discussion panel on neuroscience evidence in the courtroom - my blog coverage of the event can be read at the link.

He will talk about the ethical challenges that neuroscience raises, and what we, as scientists can do about it. Greely takes us back to 1969, what he calls the peaking of the first modern neuroscience ethics panic, during which the public came together in concern over many neuroscience themes, including neuroscience’s ability to control minds. He notes that concern over mind control led to many regulations being put in to place, based purely on speculative science. Greely fast forwards to today, where he sees the same trend towards public concern, whereas now the panic is caused by science that is actually available, as opposed to the more speculative nature of the science that was causing concern in 1969.

Greely discusses the problem with public policy being established based upon science that isn’t very good – for example eugenics programs based on our knowledge of genetics. He notes that in the case of genetics, fair and responsible public polities are well established, and the maturity of this process is about 10 years ahead of neuroscience.

What are the issues raised by neuroscience? Greely notes we are in a golden age of neuroscience, we are learning a phenomenon amount about neuroscience, and we care a great deal, both at the individual and the social level, about neuroscience given the close association between our brains and our minds. As we learn more about the brain, we will learn more about human thought and motivations. The ethical issues being raised fall into several categories. The first one of these is research ethics – as we learn more about the neuroscience, we will begin to consider questions regarding the ethics of doing research. For instance, what are the ethics regarding incidental information gathered during experiments – for instance tumors discovered during fMRI studies. Alternatively, ethical issues generated by storing brain images of participants in a database – can those images be used to advance information in ways the participants disagree with?

Greely turns to the question of how neuroscience will change our society, including changing education, medical care, and the law. Greely will talk about 6 different ways that neuroscience will impact the society – prediction, mind-reading, responsibility, consciousness, treatment and enhancement.

Prediction: neuroscience is helping us predict better things about people’s behavior. Sometimes, this involves predicting future disease states – neuroimaging or genetic predictions of who will develop Alzheimer’s. Now, this seems like a good thing, but what are the implications. If our ability to predict Alzheimer’s was coupled with a treatment, this would be fantastic. But as in the case of many genetic predictions, we often are able to predict despite being unable to prevent the occurrence of the disease. Greely notes that we are protected under federal law from discrimination based on genetic predispositions, but not predictions based upon brain scans. Predictive information is not just information - it has consequences, both good and bad. Greely poses the question of who will be responsible for producing the predictions (doctors, companies), and who will be able to have access to the information, beyond the patient. He wonders what we would do with information that predicted with 100% accuracy which 8/1000 children will develop schizophrenia, or make accurate predictions on future criminal/violent behavior. He states that if we can ask the question (what do we do with the information), someone will want to answer it.

Mind-Reading: Greely repeats a line I have heard from him before, that humans are all mind-readers. It is important for us to figure out what those around us are thinking, generally using facial cues, body language, etc… He comment that we all try to do it, but we just aren’t very good at it, and the world would look a lot different if we were better at it. And with neuroscience, we are getting better at it. There are many examples of imaging research where scientists look at activity and make suppositions about what the subjects are subjectively thinking. Now, much of that research involves figuring out whether, for example, a person is thinking of a place or a face – this is not of immediate applicability in the courtroom. But what is applicable is research that is attempting to figure out what people believe or think: e.g. lie detection, figuring out whether people are actually feeling chronic pain, whether people are biased. He introduces the current two commercial companies that offer lie detection, and the two recent court cases that asked whether they would allow fMRI-based lie detection as evidence (they both said no). Greely notes that there is currently no regulation of this field but people are still applying the technology.

Responsibility: Greely discusses recent court cases where the defendants use neuroscience brain scans to claim insanity. A more common argument in these court cases is that its not the defendants fault, it is the fault of their brain and how it works. What will juries do when told that a defendant is a psychopath, and their brain makes them a murder?

Consciousness: Greely brings up the recent paper where two groups showed that of a group of 54 patients diagnosed as being in a vegetative or minimally-conscious state, fMRI scans showed that in 5 patients, being told to plan a motor task resulted in activity in the motor planning area. In addition, 4 patients showed activity in brain regions responsible for navigation when told to imagine walking through their homes. Finally, they took one patient, who had been diagnosed in a vegetative state for 5 years, and showed that he was able to answer personal questions by selectively activating either the motor planning or navigation area. What will we do with that information? Greely comments that doctors at Stanford have already started having families of patients diagnosed as being in a vegetative state ask that the patient be put into an fMRI scanner.

Treatment: Greely wonders what happens when we learn how to “cure” things that are not diseases, such as “deviant sexual behaviors”? What happens when a neuroscience attempts to cure addition with brain lesions, as happened recently in China, where doctors made electrolytic lesions of the nucleus accumbens of soldiers addicted to opium. They reported that after the lesion, soldiers did not crave opium, but Greely notes that the peer-reviewed paper did not report what else the soldiers did not crave. Another example are laws requiring that people convicted of a long list of sexual offenses are required to undergo chemical castration, despite the fact that we don’t have much information regarding the efficacy or safety of the chemicals used for the castration. In addition, even if we know that treatments for addition, psychological diseases, etc… are efficacious and safe, when do we mandate their use?

Enhancement: Many (most) scientists use mind-altering drugs – caffeine and chocolate both alter brain chemistry. But there are greater numbers of students who are now using Ritalin without a prescription to enhance their cognitive abilities. Of course, Ritalin and other drugs like it are not that good at enhancing cognition. But what about memory-enhancing drugs developed to treat diseases such as dementia and Alzheimer’s? What do we do if these drugs work on 20-year olds? What should universities or medical schools do about the availability of these drugs? Greely states emphatically that the single greatest cognitive enhancer is primary education, the ability to read and write. What did we do about it? We made it mandatory. How will be respond to a new host of drugs.

And lastly, Greely turns to the question of how society will respond to neuroscience research regarding the human condition. How will we assimilate information regarding the differences (or lack thereof) between the brains of humans and other animals? What about consciousness – when we can identify it, how will this alter how we treat patients, or fetuses? What about free will – how society react once we can identify the exact mechanisms that lead to our decisions, when we can show that circuits in our brains have made a decision long before we consciously acknowledge that decision. How will religion be affected? Greely imagines that it won’t affect society too much – the general public will continue to believe in free will not matter what evidence neuroscience throws at them.

Having talked about these issues, Greely turns to how neuroscience should start to handle them. The first step is to conduct research to show conclusively whether the techniques mentioned over the past hour are effective and safe. Going further, are questions about how we use these techniques if they are effective and safe? Neuroscientists have perhaps a smaller role, but an important one in making sure the public is aware of the complexities of the science and the techniques. And lastly, the deeper existential questions – and here Greely states that neuroscientists and non-neuroscientists all are on an equal footing, each with something to contribute.

So what can we, as neuroscientists, do? Greely calls us to consider the ethical issues of our own work, and to talk about these ethical issues, whether they come out of our own work or the work of others. He encourages neuroscientists to get involved, to join the Society for Neuroethics, to communicate with the public on these issues, to bring our sophisticated understanding of the strengths, weaknesses and limitations of neuroscience to discussions in the public domain. Greely tells us that he must believe that the more we discuss these ethical issues, the less likely we are to mess up the big decisions. In conclusion, he hopes he made us think about the short and long term implications for neuroscience on society, and that he had convinced us of the critical need for educated neuroscientists go get involved in the introduction of our knowledge into society.

Note: Greely suggests that those interested in asking him questions should email him at hgreely@stanford.edu.

SFN: Wurtz and Brain Circuits for Active Vision

Gruber Lecture: Brain Circuits for Active Vision. Robert Wurtz, PhD, National Eye Institute/National Institutes of Health After an brief introduction to the Gruber International Prize Program by Sarah Hreha, Executive Vice President of the Peter and Patricia Gruber Foundation, Michael Goldberg (and his excellent bow tie) present the 2010 International Research Award for Neuroscience to Laura Colgin and Jason Shepherd. Congratulations to them both.

This is followed by the presentation of the Gruber Prize to Robert Wurtz by Sten Grillner (chair of the Neuroscience selection advisory board for the Gruber Foundation). Wurtz long, auspicious career is described, highlighting his groundbreaking work on the visual system of awake-behaving primates. The official text of the award: “Honored for his pioneering work in the neurophysiology of visual cognition, which has led scientists to a deeper understanding of how the brain is organized to produce behavior.”

And after a few acknowledgements, Robert Wurtz begins his talk.

Wurtz notes the usefulness of having post-docs in the lab… for when a monkey gets loose.

When presented with a visual scene, we have the perception that we see every part of that scene with equal clarity at all times. However, neuroscience has shown that this is emphatically not true, we constantly make saccadic eye movements that focus our high acuity vision to examine different parts of the visual field at any time. These eye movements shift the fovea, and visual attention, displacing the retinal image of the visual scene and producing a blurred sweep of visual information during the saccade movement itself. And yet with all this movement and displacement, our brain computes the visual information to produce the illusion of a unified, clear visual field.

The talk will address four points: 1) an overview of the brain areas involved in active vision, 2) attention (enhancement of vision with saccades), 3) suppression (decreases of the inter-saccade blurring) and 4) the mechanism that generates saccades, which underlies both attention and suppression.

First, Wurtz describes the methodologies used to study active vision in awake monkeys. The basic setup is to record neuronal activity from head-fixed monkey who are fixating on a visual location.

Next, he outlines the basic system underlying the generation of saccadic eye movements. From V1, the pathway extends to the posterior parietal cortex and the frontal cortex, and thereon to the superior colliculus, and to the midbrain and pons for generation of motor output. To highlight the role of the superior colliculus, which is a critical component of the saccade generating circuit. For the purposes of the talk, the SC can be divided into two layers, a superficial, retinorecipient layer, and a deeper layer that encodes information regarding planned saccade movements. These visual-motor neurons display a burst of activity immediately prior to the eye movement.

With this type of activity introduced, Wurtz presents a two-fold experimental strategy: 1) correlation of neuron activity to behavior and 2) perturbation of neuron activity via activation or inactivation of neurons. He notes that in the second part of this strategy, classic techniques have been chemical or electrical means, but that recently they have turned to optogenetic inactivation of neurons located in the deep layers of the superior colliculus (this in collaboration with Ed Boyden’s lab).

Correlating and perturbing neuronal activity leads to classical view of superior colliculus, namely that it is largely involved in motor generation of the saccade. Newer research has suggested that the superior colliculus is also a major source of ascending pathways to inform the cerebral cortex about the saccade. These ascending pathways leave the superior colliculus and go directly to the thalamus before going to cortical areas (for example, SC to MD to FEF; SC to Pulvinar to LIP/MT or SC to Reticular Nucleus to thalamocortical relay neurons (LGN)).  Wurtz will focus on the SC to Pulvinar to LIP/MT pathway.

We now turn to the topic of spatial attention, specifically looking at the phenomenon of change blindness. We are showed a demonstration of change blindness, which demonstrates the power of spatial attention to direct

Wurtz describes two types of attention. The first he calls onset attention, which generates saccades to new objection (also known as bottom up, or involuntary attention) – this is eliminated during the change blindness test. The second type is goal directed attention (aka top-down attention) which remains during the change blindness test. Wurtz will discuss this second form of attention, and the role of the superior colliculus. Results from experiments demonstrated a motor theory of attention which postulates that the mechanisms generating saccades to a target contribute to a shift of attention to that target – this theory was demonstrated particularly well by Tirin Moore’s 2002 experiments in stimulating FEF to shift attention and gaze.

The question for Wurtz examined was if the superior colliculus saccade generation activity contributes to attention modulation in cortex. They used a change blindness task, modulating SC activity to attempt to alter cortical processing of visual information. He first describes the paradigm of motion detection change blindness. Briefly, the subject fixates, is presented with 3 groups of dots that are moving in a direction, with out of those groups switching directions after a blank screen is shown (to induce change blindness). Cueing the participant to which group is likely to switch will shift visual attention, increasing the ability of the participant to detect the change in direction. If the visual cue is replaced by stimulation of the superior colliculus, they see a significant improvement in the ability of monkeys to detect change in the stimulus. So stimulating a neuronal component involved in saccade preparation produces a behavioral effect similar to attention. From this research, Wurtz notes with great satisfaction a cognitive function like spatial attention can be understood at the mechanistic level of neuronal activity.

Wurtz now moves on to the suppression of visual activity that occurs during the saccadic eye movement itself. The classic explanation involves corollary discharges, where information generated in sensorimotor processing is sent as a corollary discharge to other brain areas, where it informs those areas about the movement that is about to be made. Where are the saccade corollary discharges produced? Wurtz suggest it is the superior colliculus – he notes that they have identified two corollary discharge pathways: SC to MD to frontal cortex as well as SC to inferior pulvinar to occipital/parietal cortex. To summarize the research on this first pathway: the SC to FEF path provides a corollary discharge that acts of compensate for the displacement of the image on the retina. The second pathway may contribute to elimination of the blur from saccades (aka saccadic suppression).

Why look at superior colliculus as the origin of corollary discharge for saccadic suppression? Recordings from SC neurons show reduced responses to visual stimuli during movement. Furthermore, saccadic suppression starts before the saccade, and SC neurons demonstrate a decrease in responsiveness to visual stimuli immediately prior to saccadic movement. This resulted in a hypothesis stating that intermediate SC layers receive inhibitory inputs – slice recordings support this as occurring. But how does SC-mediated saccadic suppression get into the cortex? Recordings from MT neurons demonstrate saccadic suppression similar to the suppression seen in SC neurons. But are is this cortical suppression the result of the SC suppression? One problem is that it is not clear what the actual pathway is. Wurtz describes experiments that have been done to locate the pulvinar relate in the circuit between the SC and MT. He notes that they have found some relay neurons in a subregion of the inferior pulvinar that are connected to both SC and MT (this research done by Rebecca Berman and involved recording from pulvinar neurons while stimulating both SC and MT).

So does this circuit carry saccadic suppression? To test this, Berman has been recording MT neurons that show saccadic suppression while inactivating SC – her results show that in the absence of SC activity, there is an increase in MT neuron activity – suggesting that suppression in SC contributes of saccadic suppression in cortex. Wurtz notes that MT responses are a combination of SC and V1 inputs – inactivation of the SC removes one (inhibitory) input, leaving only the excitatory V1 inputs.

[Bloggers commentary: the role of the SC-pulvinar-MT connection in suppression of MT activity should perhaps be taken with a grain of salt. The exact components underlying the suppression of MT are not as clear as Wurtz suggested. The pulvinar is not an inhibitory area, so how it could be directly suppressing MT activity is not clear. One possibility is that inhibitory circuitry within in the SC is responsible but again, the exact players involved have not been identified. And intra-SC inhibition would not support their model of MT activity being the sum of SC and V1 inputs, with knocking out SC resulting in increased activity because of removal of inhibitory drive. Indeed, that model would require pulvinar to be responsible for inhibitory drive onto MT, something that is not supported by any currently described circuitry. If intra-SC inhibition is responsible, then you would expect that inactivation of SC would yield MT activation equal to drive from V1 (aka removal of additional excitatory drive), with no additional enhancement of MT (as might be expected if SC inactivation removed inhibitory drive, which Wurtz suggested is the case). In conclusion, the exact connections and role of the superior colliculus in attention and saccadic suppression have not yet been fully defined, End of commentary.]

To sum up his talk, Wurtz restates his claim that our perception of visual stability can be understood at the level of simple neuronal circuits. In conclusion, he notes that the brain circuits he has discussed all have the same basic structure, with their different functions dependent on what signals are conveyed and where those signals are directed. He notes that this structure suggests that corollary discharges and spatial attention can be viewed as variant of a general scheme. Finally, he point out the progress being made in elucidating the brain circuits that underlie cognitive processes for active vision. Still to be fully described are the mechanisms underlying decision for action, working memory, and rewards/values. He notes that understanding these cognitive functions depends on studying circuits at the highest levels of the cerebral cortex, and that understanding of these functions is necessary for comprehending the many diseases of the brain. He lends his support for research utilizing the monkey brain as an experimental model, stating the monkey brain provides unprecedented opportunities for understanding a wide range of cognitive processes and diseases.

Dyslexia and Pattern Detection in Peripheral Visual Fields

Principles of success and failure in reading instruction via the lens of systems neuroscience. McCandliss, Sackler Inst- Weill Med. Col The basic heft of this talk was to present evidence for enhanced visual abilities in dyslexic individuals. The background for this student is two-fold. First, that the parents of many dyslexic school children has claimed for many years that their children are endowed with enhanced visual abilities of one flavor or another. Researchers have been moderately dismissive of these claims (at least according to the speaker), but recently have attempted to directly test these claims. One study showed Escher figures to individuals and quantified the speed with which the subjects could distinguish between possible and impossible structures. The results showed that dsylexics can identify/analyze Escher figures twice as fast as non-dyslexics.

Jumping off from this finding, the researchers looked at dyslexic astrophysicists. A common task in the field of astrophysics is black hole detection. This task , involves identifying patterns amongst a complex field. The researchers hypothesized that dyslexics would have heightened sensitivity for pattern detection across a wide visual field. This is in part based on previous results showing that dyslexics are less able to detect patterns in the foveal receptive fields. The results were that dyslexic astrophysicists are remarkably superior at detecting black holes, showing a significant increase in signal-to-noise threshold at periphery of visual field.

Next, the researchers expanded to wider population, asking students to detect a letter hidden in a photograph. Dyslexic students showed heightened ability to detect the hidden letter - this suggests to the researchers that dsylexics have a differently constructed visual system. The speaker concludes his talk with the comment that they have no idea where the difference is (retina versus cortex versus any other visual-associated brain region).

A question from the audience: what about neurologists who have to read EEG’s, X-rays, or fMRI. The speaker suspects that dyslexics would also be better at pattern detection in these cases than non-dyslexics.

Another question: Are the differences in eye-scanning during reading of dyslexics accounted for by the lower power of the retina to detect patterns and the concomitant increase in peripheral pattern detection capabilities? Answer: possibly, although they have not done the research.

SFN: Calcium Signaling and Alzheimer's

Presenilins, neuronal calcium signaling, and Alzheimer's disease. Presented by I. BEZPROZVANNY from UT Southwestern Med. Ctr. Dallas. This talk was part of the New Advances in Calcium Signaling in Neuronal Function and Disease Symposium. The initial question proposed by the speaker is how mutations in presenilins (PS-FAD mutations) cause excessive calcium release from the ER. They hypothesize that presenilins function as ER calcium leak channels. Mutations disrupt ER calcium leak function and result in overfilled calcium stores. Loss of function for presenilin ER calcium leak becomes gain of function.

Normally, presenilins act in homeostasis of ER calcium stores. In the KO cells, this homeostasis is disrupted, so that ER calcium stores are overfilled, and when ER calcium stores are released, a much greater concentration of calcium is released.

The initial discovery and analysis of ER calcium leak function of presenilins was based on bilayer recordings of recombinant proteins and on calcium imaging experiments with PS DKO mouse embryonic fibroblasts. One question the investigators had was whether presenilins function as neuronal ER calcium leak channels. The answer is yet – recordings (and imaging) from neurons in their presenilin-KO mouse show a 2-3 fold increases in ER calcium pools as compared to wild type neurons.

So with evidence that presnilins are neuronal ER calcium leak channels, they next wondered how neurons compensate for the lack of presenilins. They found a increase in the Ryan receptor in neurons lacking presenilins – implying that the RyanR is involved in homeostatic compensation. Mice lacking RyanRs display a significant increase in the calcium pool. Combination of the presenilin and RyanR KO shows a 15-20 fold increase in the size of the ER calcium pool – significantly enhanced compared to the 2-3 fold increases in calcium pool size that result in the individual presinilin or RyanR KO conditions.

This suggests a model where both presinilin and RyanR are involved in homeostasis of ER calcium stores. Removing one of these mechanisms shifts the load to the other mechanism. However, removal of both mechanisms results in breakdown of ER calcium pool homeostasis, leading to apoptosis and accumulation of amyloid plaques typical of Alzheimer’s.

The speaker notes that loss of ER calcium leak channel homeostasis is not the only determinant of familiar Alzheimer’s – there are several other identified mutations. So the question is, given these multiple mechanisms, is there a downstream mechanism that they all affect, disruption of which results in Alzheimers. He hypothesizes that the identified mutations that underlie familial Alzheimer’s share a tendency towards network hyperexcitability. He proposes that mutations in presinilin cause disruption of ER calcium levels (via altering levels of AB42/40), disrupting neuronal hyperexcitability, which in turn activated a positive feedback loop that ends up further increasing ER calcium stores via up-regulation of AB42/40. Note: the speaker presented a more complex picture of the protein mechanisms behind this loop, further describing a second loop that he proposes to be initiated by hyperexcitability, but spoke too fast for me to note down the specifics.

So how does this relate to aging neurons (due to the link between Alzheimers and advanced age)? He notes that aging neurons are known to have increased levels of internal calcium, and perhaps this tendency is able to initiate the multiple feedback loops he proposed.

SFN: Normalization Model of Multisensory Integration

Normalization model of multisensory integration: Tomokazu Ohshiro. et al. Many physiological studies have investigated how multisensory neurons respond to inputs from different modalities. These neurons display some particular properties that speak to how they integrate the information from multiple modalities. However, a simple computation framework that accounts of these features have not been established.

The basic model proposed by the speak is a divisive model. The basic setup is as follows: individual layers that are modality specific receive information from specific sensory structures. Multiple single-modality layers, with matching (aligned) receptive fields, then target the same multisensory layer, with the inputs from each modality summed in a weighted fashion. Output from this computation is used to divisively normalize the output of the multisensory layer.

Ohshiro next describes the principle of inverse effectiveness, one of the particular properties of multisensory neurons which have not yet been successfully modeled, but which their divisive model supports. He shows an example from cat superior colliculus: when multisensory neurons are co-presented by visual and auditory stimuli that are at threshold (optimal) intensities, the activation is greater than the sum of the responses to each stimuli alone. However, additional multisensory drive (aka increasing the intensity of the multisensory stimuli to super-threshold, non-optimal stimuli) results in a suppression of the responses, such that they are less than the sum of responses to each stimulus alone. Put another way: bimodal responses are larger than the sum of the responses to each stimuli alone at weaker stimulus intensities, but smaller than that sum at stronger intensities. Ohshiro notes that according to their model, this response suppression becomes more robust as you add more sensory types.

Ohshiro moves on to the spatial principle of multisensory integration: another property he will account for in his computational model. Again he presents an example from the superior colliculus, showing that there is a spatial computation, such that if the different inputs are offset spatially, then the bimodal response gets weaker. This suppression of the bimodal response when multisensory inputs are not spatially aligned is a critical prediction of their normalization model, and confirms that the multisensory integration mechanisms is based on aligned spatial maps of sensory inputs.*

Shown next are recordings from Macaque MSTd neurons that again demonstrate that a bimodal stimulus produces a suppression of response when the stimuli are non-optimal (highly superthreshold), whereas weak bimodal inputs are enhanced relative to the sum of responses to the individual modalities.

In summary, Ohshiro et al propose that a multisensory version of divisive normalization can account for basic empirical principles of multisensory integration. Furthermore, they present both a computational model, and physiological data, demonstrating that divisive normalization can underlie the observation that non-optimal input, which is excitatory on its own, can produce cross modal suppression.

*This should not surprise aficionados of the superior colliculus, which contains aligned visual and auditory visual maps, as well as a multisensory integration circuit (see work by Eric Knusden).

SFN: Super-Resolution Synaptic Imaging minisymposium

This was my first event of the conference, so I hope, dear readers, you will keep in mind that I was still getting my science head screwed back on as I took all this down... The session was chaired by U.V. Nägerl from the University of Bordeaux and T.A. Blanpied of U. Maryland School of Medicine. They introduced the session by outlining the critical importance of understanding the structure and function of synapses and pointing out the technological gap that still exists in our ability to investigate these nanoscopic structures. However, we are embarking on a new era in synaptic imaging with the advent of a number of new techniques that bend or break the traditional diffusion limit to image structures in the range of 10s to 100s of nanometers, making synaptic imaging a reality.

The first speaker was D.A. DiGregorio, who admitted that his lab was still working within the boundaries of the diffusion limit, mainly, he joked, to find out just how bad it is for synaptic imaging. He is focused on the giant calyx of Held compound synapse as a model in order to investigate the relationship between the mutual developmental plasticity of morphology and physiology. Specifically, his lab has produced evidence that the increased precision of firing at this synapse with age may be related to refinement of spatial coupling of vesicles and presynaptic calcium channels. His technique involves a refinement of confocal imaging, using "spot imaging" to enable sub-millisecond temporal resolution rather than the slower method of line-scanning.

I'm afraid I missed some crucial parts of the next talk, but here are a few of the main points I caught: A. Triller discussed the role of syntaxin in the docking and fusion of vesicles. His group have labeled syntaxin with pHGFP, a pH sensitive fluorescent marker which localizes to the cell surface. FRAP imaging shows the molecule to diffuse rapidly within the plasma membrane, and the lab uses this diffusion to model sub-membrane events using quantum dot tracking. One principal observation utilizing this method was that syntaxin molecules pause in their free diffusion temporarily when they pass presynaptic sites.

U.V. Nägerl then spoke about imaging actin dynamics inside spines using STED. He eulogized spines as amazing signaling machines with thousands of proteins, key for brain function in health and disease, numerous, dense, dynamic, and SMALL, then hailed the new methods arising to allow the application of physiology and biochemistry to individual synapses and spines. 2-photon imaging has been extensively used to explore the structural changes accompanying synaptic plasticity, but differentiation of distinct synaptic compartments remained a challenge, as these compartments are smaller than diffraction limited resolution. This limit, however, is being broken by super-resolution imaging down to 10-50 nm. The group utilizes STED microscopy, which involves suppressing the fluorescence evoked by a primary laser beam using a secondary annular (or donut-shaped) illumination laser of a different wavelength. This produces a central spot of fluorescence much smaller than the diffraction limit.

Nägerl has been working to use STED to image spines in live cells, and has produced preliminary observations of nicely symmetrical distributions of spine neck diameters (which confocal tended to skew towards the larger diameters it could resolve). The lab has begun to investigate synaptic actin and tubulin to discover mechanisms of "morpho-functional" plasticity. Using Lifeact, a small peptide which reversibly binds to actin within synapses, the group has visualized actin cables inside spines and dendrites, some linear and some curved, presumably "handshaking" with microtubules in the dendritic shaft. In addition, different shapes of actin are observed in the spine head, whose shape changes with plasticity along a ~100nm range. Some evidence has been uncovered of changes in spine neck diameter with chemically induced LTP, which the lab intends to follow up with more refined methods. The group hopes to develop nanoscale imaging of dynamic signals such as Ca2+, to explore the possibility of nanoscale photomanipulation / uncaging, and to combine STED bulk imaging with PALM single-particle tracking methods.

The final talk I caught from this session was by E. Jorgensen of the University of Utah, who described his groups progress developing conjugate fluorescence and electron microscopy - a feat which has stymied many great scientists for decades. His group studies synaptic transmission at the 500nm diameter NMJ of the nematode worm C. elegans. Hoping to understand the arrangement of synaptic proteins, one might wish to tag specific molecules with fluorophores, but at that scale one would only see an unresolvable blob. The resolution (so to speak) of this problem involves 4 microscopy techniques: correlative fluor-EM, STED, PALM, and 'biplane'. First, correlative fluor-EM requires a delicate balance between the conflicting optimizations required for fluorescence and electron microscopy. Sections must be mounted on coverglass and not the grids required for TEM, and both florescence and EM-scale ultrastructure must be preserved. The single nanometer scale of TEM can be sacrificed for the ~5 nm resolution of back-scatter SEM, which permits the use of flat-mounted structures, but the tension between fluorescence and ultrastructure remains. EM works best with dehydrated tissue infiltrated with oxidizing agents such as osmium at an acidic pH, whereas fluorescence requires hydrated tissue, a neutral pH, and is severely disrupted by oxidization. Remarkably, Jorgensen claims to have found a compromise which satisfies both techniques, involving embedding tissue for EM in methacrylate plastics which tolerate moderate (5%) hydration, use of K2MnO4 (potassium permanganate) instead of osmium, and tight pH control using ethanolamine.

However, correlating EM ultrastructure with regular fluorescence imaging does not resolve the problem of how to image proteins at a subcellular scale (you are still correlating a coarse blob with the fine structure), though it may be very useful for identifying labeled cells and axons. Instead, the group has used STED on 100 nm sections in C. elegans to image the subcellular distributions of histones, mitochondrial protein TOM-20, and liprin. The trouble with the STED system, Jorgensen remarked, are that 1) it can only resolve down to 60 nm, 2) it's expensive, and 3) he doesn't have one. The group also successfully used PALM (comparable to STORM, but using photoconvertable markers rather than organic dyes) with a liprin-Eos conjugate which is photoconvertible from green to red by application of a little UV light. Preliminary results using this technique to explore the localization of Actin-4 in the worm nose have suggested that the molecule is to be found in the membrane of glial sheath cells, but not in the sensory cells, as some had presumed. Still, all this conjugation proved very time consuming, which led to the development of biplane microscopy by the lab, which, as the name implies, improves on the efficiency of PALM by imaging in 2 planes at once. The system is largely computerized - as Jorgensen proclaimed, "If you still have an eyepiece, you probably still have a rotary phone - give 'em up!" This system is now commercially available as the Vutura Avalanche (and is present in the vendor stands at SFN!)

All in all, these super-resolution techniques seem to be opening up a bright new future for imaging the tiny world of the synapse!

SFN: Vocalization Minisymposium

Neural Mechanisms Underlying Vocalization in Multiple Species: A Special Focus on Parkinson’s Disease Unfortunately, I missed most (read: all) of this minisymposium due to research-relevant poster sessions. However, I did stop in for the closing remarks, hoping to pick up some interesting tidbits.

Speakers touched on the need to look at vocalizations in animal models in both normal and diseased states. The speaker calls for better integration between the multiple disease models mentioning the possibility of using vocal changes as an early predictor to disease onset, particularly in Parkinson’s, but also in diseases such as Huntington’s, ALS, autism spectrum disorders.

A brief question and answer period:

One researcher suggests bringing back the cat as a research model. The panel agrees that there are many fine animal models, but declines to comment directly on the cat as a model system.

Another question regards the difference between production of ultrasound and “normal” vocalizations. According to a panel member, ultrasonic mouse vocalizations shift their fundamental frequencies when you alter the media through which they propagate – this is not the case for audible vocalizations from the mouse. To the panel member, this suggests distinct vocal production mechanisms for audible and ultrasonic vocalizations.

Where any readers out there present for more of the session? The comments section is open to any notes you may have from the session.

SFN Poster Session: For Lovers of the Superior Colliculus

Most of my first afternoon at SFN was spent in the poster session, specifically hunting for posters regarding functionality in the superior colliculus, and its non-mammalian homolog the optic tectum. Since I work in this brain region, most of my attention has been focused on findings applicable to my own research. Without going too in depth into my own research and why I spend half an hour pondering the release of nicotine into the superficial layers of the pigeon optic tectum (thanks to Macarena Faures' poster), some brief thoughts on the posters: For aficionados of the region, there are several groups who have successfully expressed channelrhodopsin (or halorhodopsin) variants in axons of retinal ganglion cells, using this technique to selectively activate (or inactivate) visual activity into the superior colliculus. Most of the posters show the success of the expression, it seems that we will have to wait until next year for the results of any experiments made possible by these technological advances. One exception is a group that uses a channelrhodopsin variant expressed in a sub-population of retinal ganglion cells to show that connectivity between RGCs and the circuitry of the superior colliculus is in place before maturation of the retinal opsin system. It’s possible that these results are not surprising, given previous research showing that wiring of RGC axons in the superior colliculus is dependant on spontaneous RGC waves. (Someone more familiar with the temporal placement of retinal waves during development should feel free to correct me here).

To turn back to the Faures poster, out of the Marin group - their discussion brings up the possibility that the neurotransmitter identity of a particular brain region, Ipc, has been incorrectly identified. The Ipc is the avian homolog of the mammalian parabigeminal nucleus, and has been classically thought to be cholinergic, due mostly to the presence of ChAT immunoreactivity. However, recent research is apparently calling into question the assumption that the Ipc released acetylcholine - at least according to a researcher in the Marin group. I'm going to withhold my judgement (although I am deeply skeptical) and wait for the proof positive combination of recordings and histochemistry. But that the question is being posed in the first place is fascinating to me - if their concerns are valid, it will highlight the difficulty of exactly characterizing any brain area based on expression of particular proteins.

On a less scientific note: of general amusement has been watching as my PI strode through the poster session, ending up huddled with about 4 other PI's, hotly debating the function of the superior colliculus.

SFN: Genes, Photons, and Electrons, Short Course #1, Part I

Hello from San Diego and welcome to the Stanford Neuroblog! In this post, I'll be covering the first half of SfN Short Course #1: Genes, Photons, and Electrons. This workshop focused on novel techniques for probing and manipulating neural circuits and connecting structure and function. Overall, the talks were excellent and the speakers complemented each other nicely. The course nicely summarized recent progress in experimental technology. The content of the talks also conveyed a mix of excitement at how accessible previously unfathomable circuit level questions have become thanks to novel technologies and bewilderment at how complex the relationship between neural circuit structure, function, and behavior appears to be. Below are essentially summaries of what was said and presented rather than my own commentary. I have tried to minimize factual errors or misinterpretation of the speaker's remarks, but these summaries are reconstructed from my notes during the talks and are surely imperfect. Please kindly notify me of any errors or oversights in the comments section. Enjoy and stay tuned for Part II!

Opening Remarks Michael Hausser, UCL

We can roughly divide the history of scientific investigation into neural circuits into three phases. First came the romantic age, led by Ramon y Cajal, driven primarily by the Golgi stain and pure anatomical observation. Next came the classical age. Hodgkin and Huxley, Katz, Fatt, Eccles, Llinas, Rall, Bliss and others pioneered the study of neural function at the cellular and synaptic level, while Hubel and Wiesel, O'Keefe, Georgeopolous, Newsome, Shadlen opened the doors to modern day systems neuroscience. Despite great advances, what's largely missing from this "Classical Age"? Genetic identity of cell types, recording of activation patterns of all neurons and synapse relevant to behavior, complete descriptions of underlying connectivity patterns, and the ability to clearly demonstrate causal relationships.

Now, according to Hausser, we enter the "age of enlightenment," hinting at a pun on optogenetics with an image of a mouse sporting a head mounted fiber optic glowing blue. In this age, we will enjoy a new array of technologies that he's divided into three overarching categories, following the title of the short course.

Genes

• sequencing of entire genomes
• genetic model systems
• transgenic animals and viral approaches
• genetically encoded labels / probes

Photons

• 2 photon
• super-resolution
• optogenetics

Electrons

• Dendritic patch clamp recording
• in vivo patch clamp recording
• High density arrays and optrodes

So equipped with these dream tools, what would be the dream experiment? Hausser lists a few suggestions: reconstruct the connectivity of entire circuit, measure the activity in all neurons during behavior, sway decisions and recall memories by manipulating neurons, etc.

Semi-Automated Reconstruction of Neural Processes from Large Numbers of Fluorescence Images Jeff Lichtman, Harvard

Jeff opens with a definition of the word connectomics from the OED circa 2015:

connectomics: noun plural but singular in construction

A branch of biotech concerned with applying techniques of computer-assisted image acquisition and analysis to structural mapping of sets of neural circuits or to the complete nervous system of selected organisms using high-speed methods with organizing the results in databases, and with applications of the data (as in neurology or fundamental neuroscience) - compare proteomics or genomics,

see also connectome

He then describes a related array of efforts and research directions that are commonly associated with connectomics:

• Human connectome, led by Olaf Sporns at Indiana University, to map axon projection pathways with DTI. Perhaps this should be referred to as a projectome?
• Testing Peters rule: expected number of connections proportional to product of their dendritic and axonal tree densities
• Investigation of neurogeometry and potential synaptic connectivity [Stepanyants and Chklovskii, TINS 2005]
• Blue brain project, led by Henry Markram, EPFL
• "Cajal 2.0", first pass connectome: including efforts by the Allen Brain institute, Partha Mitra Brain architecture project, fly optical project at JFRC
• Sparse labeling / reconstruction: micro-optical sectioning tomography [Li, Science 2010]. Reconstructing subsets of cells using automated/semi-automated analysis of fluorescence images. Brainbow.
• Dense reconstruction: Denk, Heidelberg, Seung. This involves dense reconstruction of neuropil structures from stacks of EM images. A great deal of  automation and quality control required from tissue handling, image acquisition, image alignment, segmentation, reconstruction, verification, annotation, etc.

Lichtman's lab is primarily involved in these last two efforts: sparse labeling and dense reconstruction. One tool useful for sparse labeling is the array of Thy1-XFP mouse lines which provide expression of a particular flurophore (CFP, YFP, GFP, etc.) in an apparently random susbset of neurons in the brain. This "random" expression results presumably due to random insertion of the transgene into genome.

He demonstrates the power of this technique by showing a series of confocal images of the neuromuscular junction of a Thy1-YFP mouse. The NMJ is innervated by YFP and AChR expressing motoneurons, and it is clear from the images that each NMJ is innervated by only one axon, thought the nerve bundle possess many axon fibers.

In the spirit of the short course having an educational component, Jeff pauses to offer tips on taking a proper confocal image.     When performed correctly, confocal offers enhanced contrast, optical sectioning, and a resolution improvement by sqrt(2) over the widefield diffraction limit. However, it is easy to saturate the fluorophores by turning the laser power too high, resulting in disproportionate out of focus signal reaching the detector. Additionally, he asserts it is important to image using the full dynamic range of the sensor (meaning few pixels lying at either end of the histogram range. This allows for lossless imaging and better reconstruction, and deliberate saturation can almost amount to scientific fraud by "throwing away" outlying pixels.

He then turns to the task of segmenting and tracing fluorescence labeled axons in confocal image stacks. The details of the algorithm are mentioned in Lu et al. 2009. Quickly, he demonstrates structural polymorphism present in left vs. right versions of same nerve bundle projecting to muscle. He also points out that individual axon paths demonstrate numerous suboptimalities, exhibiting wasted loops and back-tracking, as well as clear violations of Peter's rule.

Next up, Brainbow! Brainbow is a technique for achieving unique labeling of individual cells by combining random amounts of three fluorophores (mCherry, eYFP, Cerulean) in each cell, achieving the same effect as a TV screen combining RGB intensities to create a particular hue. The construct, which has the form thy1-lox-lox-mCherry-lox-eYFP-lox-Cerulean, uses the stochasticity of Cre splicing to achieve this random expression. Because each neuron has a unique and consistent color (defined by relative levels of red, green, and blue expression), this eliminates the need to trace axons/dendrites since there is a 1:1 correlation of color intensities at both ends of the neuron. However, if the labeling becomes too dense, the fibers in neuropil can become too thin or too weakly expressing for reconstruction.

Another side issue is how to visually display a connectome once you have obtained it. He presents a number of display formats, motivated by graph theory. The point is that there a number of possible choices, but it's clear that there are non-random features evident in the connectivity matrices observed even for small numbers of neurons reconstructed.

Lastly, he discusses the technology behind ATLUM (Automated Tape-Collecting Lathe Ultramicrotome) which automates the slicing and handling of thin brain slices embedded in plastic resin. He shows a video by Daniel Berger in Sebastian Seung's lab which opens from a photo of a silicon plate held by a lab member on which tissue has been mounted for EM. We then gradually zoom in to the point where we see individual vesicles in a presynaptic bouton.

Imaging Neural Activity in Worms, Flies, and Mice with Improved GCaMP Calcium Indicators Loren Looger, HHMI Janelia Farm

Loren opens with the point that neural circuits underlie behavior, where a circuit is defined as a collection of neurons, their chemical identity in terms of neurotransmitters released on postysynaptic targets, their connectivity graph, the sign of their connectivity, as well as changes over time of these properties as a function of development and experience. He asserts that these properties of neural circuits are essential to making in progress in understanding circuit function and the structure/function relationship, noting that very little insight has been extracted from the complete connectome for C. elegans completed some decades back. He notes that 2 maybe 3 key points of understanding may have been derived, but mostly in the last few years.

He then borrows analogous terminology from forward and reverse genetics to describe the types of optogenetic research that are now possible. Forward optogenetics is observing neural activity optically during behavior, e.g. calcium imaging in head fixed mice on a floating spherical treadmill a la David Tank's lab. Reverse optogenetics is perturbing neural activity in order to determine causal influences of circuit elements on behavior, e.g. a mouse running in circles subsequent to ChR2 activation via head mounted fiber optic a la Karl Deisseroth's lab.

Circuits are ultimately the minimal level at which to study certain interesting behaviors, but molecules compose a circuit. Specifically, a researcher can utilize molecular probes in order to observe and quantify neural function. Loren's lab had previously solved the crystal structure of GCaMP2, a genetically encoded calcium sensor created by placing calmodulin, a calcium binding protein, inside GFP. His lab then gradually engineered an enhanced GCaMP3 by screening point mutations in GCaMP2. He briefly compares GCaMP to FRET based sensors, noting that GCaMP avoids photostability problems common with FRET because the fluorophore is not exposed and therefore not bleachable in the off-state. He briefly mentions the ongoing development of GCaMP5, which touts better SNR and faster off kinetics for enhancing the ability to distinguish single action potentials.

He then mentions an array of calcium indicators spliced to other flurophores, opening the door to spectrally separation in activity reporters, e.g. RCaMP via mRuby with a bimodal 2 photon excitation spectrum (750 and 1125 nm), CyCaMP, BCaMP, etc.

Another direction for enhancing these reporters is in achieving subcellular targeting specificity. For example, by restricting the calcium indicator within neuron nuclei, the overall signal has half the intensity and half the speed of regular GCaMP, but with the advantage of much lower background fluorescence from processes. This clean separation of glowing nuclei greatly facilitates segmentation of individual cellular signals, a direction his lab is pursuing in collaboration with Daniel Dombeck and David Tank at Princeton.

Bioluminescence as a Tool to Monitor Neural Activity in Freely Behaving Animals Florian Engert, Harvard

Florian opens with an introduction of his favorite model organism, the larval zebrafish. Specifically he employs the nacre mutant, which is perfectly translucent except for eye pigment. This translucency facilitates for a range of imaging and potentially optogenetic techniques. He employs transgenic fish which express GCaMP3 in every neuron, allowing imaging of calcium transients of the entire system. He asks the crowd for suggestions on what to call this kind of dataset: an activitome?

He states that our ultimate goal is to characterize how the brain produces behavior. The experimental desire for behaving animals clashes with the stability demands of high resolution optical imaging. The trend towards head-fixed imaging with an animal performing a task in virtual reality, as David Tank has demonstrated in mice and others previously in flies, is probably the best solution, if you can reproduce the behavior sufficiently well while tethered.

Another approach is to utilize bioluminescence. Equipping bioluminescent apoaequorin protein with GFP across a calcium binding linker creates a FRET pairing interaction, effectively creating an illumination-free calcium reporter. The approach requires the cofactor CLNZ, which is itself fluorsecent, facilitating quantification of loading efficiency. This sacrifices all spatial resolution of the imaging, as photons are usually detected and counted by a wide angle of entry photomultiplier tube. The technique's advantage lies in restoring the fish's full motility. Resolution has to be reintroduced using the specificity of the expression of the apoaequorin, presumably genetically.

He demonstrates one example of this technique by targeting the apoaequorin to the fish hypocretin system, a network consisting of 8 neurons on each side. Two types of neuroluminescence events are clearly distinguished, though both are correlated with bursts of locomotion. "Large" luminescence transients are related to long latency, short travel swims, whereas "small" events are related to shorter latency, longer travel swims.

Typically bioluminscence imaging requires complete darkness, which conflicts with the need for visible wavelength visual stimulation to elicit and modulate fish swimming behavior. Spectral separation wasn't sufficient to recover good signal, so the lab developed an imaging approach relying on temporal separation of the visual stimulus and bioluminescent photon detection. The essence is to strobe visual stimuli at a frequency well beyond flicker fusion (e.g. 1 kHz), then gate the PMT closed during times when visual light is on.

SFN: Saturday's Blogging Schedule

Welcome to a special SFN section I'm going to call: Where's the Neuroblogger?

For the reader's who are holding their breaths in anticipation of our SFN blog posts*, each day I will post a list of that day's events that I may attend (final attendance choice to be decided as I go along). Currently, the plan is to live-blog talks here at the Neuroblog, and to tweet about posters and other nonsense (via our twitter handle @stanfordneuro).**

Without further ado, here's a list of the Saturday events***:

1-3 pm: Modulation of Synaptic Processing Nanosymposium, Room 25A

1-4 pm: Neural Basis of Auditory Perception and Action Nanosymposium, Room 33C

1-5 pm: Poster Sessions, including: Nicotinic AChRs, Oscillations and Networks, Saccades (Superior Colliculus, Brainstem and Behavior)

2-3 pm: Architecture, Symmetry, and Mechanism of Ionotropic Glutamate Receptors, Ballroom 20.

5:15-6:25 pm Adventures in Non-translational Research: Neuronal Differentiation and Mechanosensory Transduction in C. elegans, Ballroom 20.

*G. Panagiotakos, this means you.

**For those of you who refuse to use twitter, never fear. I will compile our best tweets (and those of fellow SFN10 twitter users) in a blog-reader-friendly post.

***I'm not arriving at SFN10 until Saturday morning, so will not be blogging any of the events that occurred on Friday.