Shea Lab Journal Club: Monitoring large populations of locus coeruleus neurons reveals the non-global nature of the norepinephrine neuromodulatory system

Monitoring large populations of locus coeruleus neurons reveals the non-global nature of the norepinephrine neuromodulatory system (2017) Totah et al. http://biorxiv.org/content/early/2017/02/18/109710

This is the latest installment of  an ongoing series of capsule summaries of the Shea Lab journal club meetings. We are doing this to actively provoke open discussion of papers we read that are either published in a traditional journal or BioRxiv.

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The brainstem nucleus locus coeruleus (LC) is nearly the sole source of the neuromodulator noradrenaline (NA) for the forebrain. The relatively small number of cells in locus coeruleus collectively broadly and diffusely innervate most of the forebrain. This projection pattern and the seemingly homogenous cellular composition of LC has inspired the conventional view that LC output carries a unitary signal, or some sort of global variable, to all brain regions.

Some recent anatomical data is leading researchers to reexamine that model. For example, this paper from the Waterhouse lab and this one  from my colleagues Justus Kebschull and Tony Zador show that there is heterogeneity in LC, and that individual cells may target a more restricted brain location than was previously appreciated. This potentially provides an anatomical substrate for LC to differentially modulate target brain structures. However, this would still depend on cells targeting those structure carrying distinct functional activity patterns.

The fundamental question that this paper tries to answer is: “To what extent do different LC neurons carry correlated or distinct information?” This is a very important question that has implications for past and ongoing work in my lab involving LC and NA, so with great interest, I selected the bioRxiv preprint for discussion in a recent Shea lab journal club. The paper is very complex and contains a lot of analysis, but my intention is not to write a comprehensive review. Here I just want to comment on some what I see as some important limitations of the study as written. Also, since this paper is in the preprint stage, maybe my thoughts will be useful to the authors moving forward.

In any neural system, there are always processes occurring in parallel at many time scales. For example, very rapid firing rate fluctuations may closely track the fine temporal structure of a vibratory tactile stimulus. Or auditory system neurons may “phase lock” to the periodic structure of sound. On the other hand, some neurons vary their spiking output dramatically across the circadian cycle of sleep and wakefulness, a much slower pattern. This contrast is also found in the multiplexing of firing rate fluctuations at long and short time scales in LC neurons.

For decades, researchers have recognized that LC neuronal spiking patterns are composed of slowly varying “tonic” spiking patterns that are punctuated by “bursts” of spikes at irregular intervals. Tonic firing changes occur over minutes or more, and individual phasic bursts last several hundred milliseconds. I would argue that these observations point to a range of time scales over which LC firing is likely to change with respect to behavior. As a result, I was disappointed that the authors of this paper performed the majority of their analyses at time scales that are shorter than that.

In many ways, the analysis in this paper makes sense. When one is looking at neuronal correlations, typically they are seen on a spike to spike basis. If cell B’s spikes are reliably occurring with a short delay after each of  cell A’s spikes, then that shows that A and B are correlated. The presence of gap junctions between some cells in LC makes these correlations largely expected. At this timescale, the authors found fewer correlations than one might have thought. The authors also looked for correlations over longer 200 ms windows, and found surprisingly few.

So why did this paper fall short of fulfilling my initial enthusiasm? It’s because the authors for the most part didn’t examine the relationship between firing in different cells over the timescale that I suspect is most closely related to behavior. To me, the relevant questions are: What is the relationship between phasic bursts across cells? Do they occur in a synchronous, coordinated manner? As tonic firing rates rise and fall over minutes within a single cell, does firing in other cells rise and fall in a coordinated manner? The authors quantified correlated firing over a maximum window of 200 ms, which is barely enough to encompass one of the phasic bursts. They did examine longer timescales, but only periodic activity; that analysis was not sensitive to slow, aperiodic fluctuations. So, the analysis demonstrates a surprising lack of spike to spike correlations, but they didn’t answer any of the questions above.

I imagine they may have the data that could answer these questions, and I would be very interested to know the results. To best answer to these issues however, one needs to fully explore a broad range of firing rates. That would be best achieved in an awake animal experiencing a range of brain states, but these experiments were performed in anesthetized rats. I don’t know whether they saw large swings in tonic firing rates and frequent phasic bursts under these conditions. In the spirit of constructive feedback, since this is a preprint, I recommend to the authors to describe how firing rates and bursts are coordinated over longer times. Otherwise, congratulations to them on a nice piece of work!

 

Shea Lab Journal Club: SHANK3 controls maturation of social reward circuits in the VTA

SHANK3 controls maturation of social reward circuits in the VTA. Bariselli et al. (2016) Nature Neuroscience doi:10.1038/nn.4319

This is the latest installment of  an ongoing series of capsule summaries of the Shea Lab journal club meetings. We are doing this to actively provoke open discussion of papers we read that are either published in a traditional journal or BioRxiv.

This meeting was attended by members of the Shea and Tollkuhn labs.

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Autism spectrum disorders (ASD) are typically marked by disinterest in social engagement. It is widely accepted that mouse models of ASD also commonly show reduced interest in social partners. Neuroscientists who study ASD have a very limited understanding of the neural circuit basis for social disinterest. However, these authors speculate that social interaction may activate brain reward pathways overlapping with those that are activated by other types of reward such as sucrose or drugs abuse. Moreover, they suggest that mutations that cause ASD may alter social behavior by interfering with the function of these pathways. Specifically, this paper focuses on the role of dopamine releasing neurons in the ventral tegmental area (VTA). As someone who’s interested in this topic, I think this is a reasonable and interesting speculation worth investigating.

To test this idea, the authors use viral delivery of shRNA to knock down Shank3 expression in VTA neurons. Shank3 is a scaffolding protein that contributes to the organization of the postsynaptic membrane, and mutations of Shank3 cause Phelan-McDermid syndrome and other ASDs. By comparing properties of these manipulated neurons to neurons in control mice, they show altered synaptic properties and in vivo spontaneous activity. Mice with shank three knockdown in the VTA also show moderate but significant changes in their preference to approach an enclosure containing a social partner versus an empty enclosure. This behavior phenotype may be related to electrophysiological changes they see in VTA neurons, but it is unclear.

Importantly, in Figure 8 the authors attempt to restore preference for social interaction in Shank3 knockdown mice by optogenetically boosting activity in dopamine releasing VTA neurons. Ostensibly, this experiment makes a more direct link between VTA neuron function and sociability. Most of our journal club group was deeply confused by this experiment. According to the manuscript:

“In vivo, we stimulated VTA DA neurons³⁵ during T₂ when mice were in close proximity to the social stimulus (Fig. 8e). ShShank3 mice that did not receive optogenetic stimulation showed a reduction in social preference at T₂ (Fig. 8f,g). However, phasic stimulation of VTA DA neurons increased social preference during T₂ in both scrShank3 and shShank3 mice (Fig. 8f) and increased normalized social preference of shShank3 mice to the levels of the scrShank3 control group (Fig. 8g).”

So if I understand correctly, the experimenters gave the mice an extremely pleasurable blast of dopamine every time they approach the social partner. Perhaps not surprisingly,  all of the mice started hanging around the social partner, irrespective of whether they had Shank3 knockdown. Are we missing something? How is this different from intracranial self stimulation? Aren’t they just conditioning the mice with the reward signal? I’m curious whether this came up in review. For example, did anyone suggest they try stimulating dopamine release when the mouse approach the empty closure?

I welcome comments from readers and the authors. Maybe there’s something I don’t understand.

 

Fitter, Happier

“See if you can fit it on the paper
See if you can get it on the paper”

 

There were several discussions of scientific “productivity” on Twitter yesterday. It’s long been clear to me that people have wildly different ideas about what this means and how to measure it. Many times you find people talking about how many papers a scientist has published, but does anyone seriously think that that is a useful number? One major factor is that individual researchers and communities have dramatically different ideas about what constitutes a publication unit. I remember being very annoyed when my first grant, which was directly based on my postdoctoral work, was reviewed with a ding that it was based on “a single publication.” Setting aside the fact that I didn’t invent a whole field and there was long literature preceding me, why is that in and of itself a neg? That was four years of work done entirely by me. I probably could have portioned out some number of smaller nuggets and published them separately, but why is that a good thing?

So I was interested in this exchange that came in a larger discussion of standards for review of NIH grants:

In a strict sense, Drug Monkey is right because science is never complete, but his argument is really a straw man. We can’t pretend that all papers are anything close to equal in terms of scientific productivity. And to head off an inevitable response, I am not talking about Glam. I am also not talking about middle vs. first author papers. It is absolutely the case that first author papers can reflect a wide range of what we deem to be productivity. In my opinion, at the extreme that range may even plausibly span an order of magnitude.

My attitude is that it is more efficient and better for science to publish your data in larger chunks, but I understand that many people feel differently. I’m interested in hearing from people in the comments. Given the same data, what is the argument for splitting it up? How do you know when to stop and publish something?

 

We Jam Econo

In 1985, punk band The Minutemen released an album entitled Project: Mersh, which was a self-conscious, tongue-in-cheek attempt to make a record that was marketable without necessarily bothering to make it any good.

The Minutemen were to say the least peculiarly idiosyncratic characters and their lyrics and interviews were peppered with their own insider lingo. For example, “jamming econo” referred to their preference to operate cheaply as a band, and their landmark record “Double Nickels on the Dime” was so titled in mockery of Sammy Hagar’s cheesy declaration “I Can’t Drive 55.” As I understand it, they felt Hagar sadly needed to prove he was wild somehow since he was too cowardly and/or lacking in imagination to be wild musically. Having no such hangups, The Minutemen proudly drove “Double Nickels on the Dime.”

“Mersh” was their term for “commercialism” in music: formulaic in approach, superficially alluring and ultimately hollow. In his wonderful book Our Band Could Be Your Life, author Michael Azerrad explains it this way:

“By mimicking the ‘mersh’ form and yet destined to sell few records, they were making a point about music  biz chicanery: Any band could sound like this if they had enough money, but that wouldn’t mean they were any good.”

I’m sure you’re wondering what the point of all this is.

I am inspired to tell this story about The Minutemen because of my increasing impression that there is a convergent formula for a segment of Glam neuroscience that fits well with my understanding of what it means to be mersh. I’m not going to single out examples, but feel free to do so in the comments! To me it is typified by the kind of study that has many authors from multiple labs, with each one contributing one or two panels. Such papers often do this apparently to create the illusion of a “comprehensive” and “mechanistic” “story.” Unfortunately, they also more than occasionally rely on a logical framework wherein putting two observations next to one another means they are related. Yet these papers have appeal and get lots of attention.

In my ongoing mission to port the logic and language of punk rock to science, I propose that these papers henceforth be derided as “mersh.” Neuroscience needs more Minutemen and less Sammy Hagar.

Shea Lab Journal Club: A distributed network for social cognition enriched for oxytocin receptors

A Distributed Network for Social Cognition Enriched for Oxytocin Receptors. Mitre et al. (2016) J Neurosci 26(8):2517

This is the first installment of what will be an ongoing series of capsule summaries of the Shea Lab journal club meetings. We are doing this to actively provoke open discussion of papers we read that are either published in a traditional journal or BioRxiv.

This meeting was attended by members of the Shea and Tollkuhn labs. Names have been changed to protect the opinionated 🙂

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Dang, this is one big, hairy beast of a paper! It starts out with a pretty straightforward and very important goal: to develop a sensitive and selective antibody to the mouse receptor for the neuromodulator oxytocin and identify the pattern of expression at the regional circuit and sub cellular levels. The paper does that rather nicely and then goes on to incorporate EM, RNAseq, and in vitro and in vivo electrophysiology over the course of 13 figures.

Like I said, the first half of the paper is a tight and thorough execution of the goal as formulated above. The authors identify for the first time the network of brain regions that express this receptor, and they observe a provocative pattern of presynaptic expression that was (to me anyway) unexpected. They achieve this by bearing down on the formidable task of developing and validating an OXTR antibody

The second half of the paper runs through an series of exciting but somewhat preliminary observations primarily using in vitro and in vivo electrophysiology. Most of this stuff is super interesting, examining oxytocin’s acute neuromodulatory effects and its effects on synaptic plasticity. The down side is that many of these effects are not deeply explored, so I hope that they get followed up on. A little bird told me that a lot of these things were responses to reviewer comments, which is weird to me. It seems to me that the physiology would be better served in its own venue. Greedy reviewers!

Strengths/Praise: All present agreed that the paper’s greatest contribution was the development of a high quality antibody for oxytocin and the visualization of the brain-wide expression pattern. The apparent rigor with which this was done is a major strength. These resources provide an important substrate for future work.

Secondary, but also important, is the fact that this paper avoids the media glam image of oxytocin as a “love molecule” and takes it seriously as a modulator of neuronal activity and plasticity.

There were other nuggets of interest. The motif of presynaptic expression suggests some interesting unexpected functions for oxytocin. Also, the fact that OXT+ fibers overlapped the OXTR+ cells in certain hypothalamic regions but not elsewhere suggested to me distinct, circuit-specific modes of point-to-point and volume transmission. But that’s probably speculative of me.

Weaknesses/Criticism: Our major criticism was that the paper could have ended after 7 or 8 figures and started a new paper. But like I said, I subsequently learned that these were things the reviewers asked about, so maybe we shouldn’t fault the authors.

One other minor critique that came up was that the authors may have been a bit chauvinistic in choosing the regions they focused on. The auditory cortex is not one of the top expressing regions based on raw cell count, The counter argument I suppose is that with this kind of staining, sparse does not always imply weak. Also, looking at the  auditory cortex is well motivated by a recent high profile study by this group.

Coordination of brain wide activity dynamics by dopaminergic neurons

http://biorxiv.org/content/early/2016/03/02/042168

Garret Stuber, Heather Decot, Vijay Namboodiri, Wei Gao, Jenna McHenry, Josh Jennings, Sung-Ho Lee, Pranish Kantak, Yu-Chieh Jill Kao, Manamita Das, Ilana Witten, Karl Deisseroth, Yen-Yu Ian Shih

 

Several neuropsychiatric conditions, such as addiction, schizophrenia, and depression may arise in part from dysregulated activity of ventral tegmental area dopaminergic (THVTA) neurons, as well as from more global maladaptation in neurocircuit function. However, whether THVTA activity affects large-scale brain-wide function remains unknown. Here, we selectively activated THVTA neurons in transgenic rats and measured resulting changes in whole-brain activity using stimulus-evoked functional magnetic resonance imaging (fMRI). Selective optogenetic stimulation of THVTA neurons not only enhanced cerebral blood volume (CBV) signals in striatal target regions in a dopamine receptor dependent fashion, but also engaged many additional anatomically defined regions throughout the brain. In addition, repeated pairing of THVTA neuronal activity with forepaw stimulation, produced an expanded brain-wide sensory representation. These data suggest that modulation of THVTA neurons can impact brain dynamics across many distributed anatomically distinct regions, even those that receive little to no direct THVTA input.

Evidence for selective attention in the insect brain

http://biorxiv.org/content/early/2016/02/29/041889

Benjamin L de Bivort, Bruno van Swinderen

The capacity for selective attention appears to be required for any animal responding to an environment containing multiple objects, although this has been difficult to study in smaller animals such as insects. Clear operational characteristics of attention however make study of this crucial brain function accessible to any animal model. Whereas earlier approaches have relied on freely behaving paradigms placed in an ecologically relevant context, recent tethered preparations have focused on brain imaging and electrophysiology in virtual reality environments. Insight into brain activity during attention-like behavior has revealed key elements of attention in the insect brain. Surprisingly, a variety of brain structures appear to be involved, suggesting that even in the smallest brains attention might involve widespread coordination of neural activity.

Sensitive red protein calcium indicators for imaging neural activity

http://biorxiv.org/content/early/2016/02/29/041780

Hod Dana, Boaz Mohar, Yi Sun, Sujatha Narayan, Andrew Gordus, Jeremy P Hasseman, Getahun Tsegaye, Graham T Holt, Amy Hu, Deepika Walpita, Ronak Patel, John J Macklin, Cornelia I Bargmann, Misha B Ahrens, Eric R Schreiter, Vivek Jayaraman, Loren L Looger, Karel Svoboda, Douglas S Kim

Genetically encoded calcium indicators (GECIs) allow measurement of activity in large populations of neurons and in small neuronal compartments, over times of milliseconds to months. Although GFP-based GECIs are widely used for in vivo neurophysiology, GECIs with red-shifted excitation and emission spectra have advantages for in vivo imaging because of reduced scattering and absorption in tissue, and a consequent reduction in phototoxicity. However, current red GECIs are inferior to the state-of-the-art GFP-based GCaMP6 indicators for detecting and quantifying neural activity. Here we present improved red GECIs based on mRuby (jRCaMP1a, b) and mApple (jRGECO1a), with sensitivity comparable to GCaMP6. We characterized the performance of the new red GECIs in cultured neurons and in mouse, Drosophila, zebrafish and C. elegans in vivo. Red GECIs facilitate deep-tissue imaging, dual-color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging.

A probability distribution over latent causes in the orbitofrontal cortex

http://biorxiv.org/content/early/2016/02/29/041749

Stephanie C.Y. Chan, Yael NIv, Kenneth A. Norman

The orbitofrontal cortex (OFC) has been implicated in both the representation of “state”, in studies of reinforcement learning and decision making, and also in the representation of “schemas”, in studies of episodic memory. Both of these cognitive constructs require a similar inference about the underlying situation or “latent cause” that generates our observations at any given time. The statistically optimal solution to this inference problem is to use Bayes rule to compute a posterior probability distribution over latent causes. To test whether such a posterior probability distribution is represented in the OFC, we tasked human participants with inferring a probability distribution over four possible latent causes, based on their observations. Using fMRI pattern similarity analyses, we found that BOLD activity in OFC is best explained as representing the (log-transformed) posterior distribution over latent causes. Furthermore, this pattern explained OFC activity better than other task-relevant alternatives such as the most probable latent cause, the most recent observation, or the uncertainty over latent causes.

Conformational dynamics of Tau in the cell quantified by an intramolecular FRET biosensor in physiological and pathological context

http://biorxiv.org/content/early/2016/02/29/041756

Cristina Di Primio, Valentina Quercioli, Giacomo Siano, Branislav Kovacech, Michal Novak, Antonino Cattaneo

Impaired interactions of Tau protein with microtubules (MT) and Tau misfolding play a key role in Alzheimer disease (AD) and other neurodegenerative diseases collectively named Tauopathies. However, little is known about the molecular conformational changes that underlie Tau misfolding and aggregation in pathological conditions, due to the difficulty of studying structural aspects of this intrinsically unfolded protein, particularly in the context of living cells. Here we developed a new Conformational-Sensitive Tau sensor (CST), based on human Tau full length protein, to investigate the changes in 3D conformation and aggregation state of Tau upon modulation of its interactions with MTs in living cells, in physiological and pathological conditions. After showing that the CST fully preserves functional Tau activities in living cells, we demonstrated that MT-bound Tau displays a loop-like conformation, while soluble Tau assumes a relaxed conformation. The imaging readout based on CST allowed to discover new conformational properties of full length Tau in living cells, when challenged with Alzheimer-relevant seeds from different sources, and to learn about different ways to induce the self-aggregation of full length Tau in cells. Furthermore, it allowed to investigate the contribution to the pathology of point mutations known to alter Tau/MTs interaction.