Philosophers like Kierkegaard and Heidegger, concerned with human freedom, believed that free will decisions have a strong impact on a person and that the process of realizing our freedom and accepting agency for the actions in which we manifests ourselves is rather long and emotionally pretty painful (despair, anxiety). Non of the designs of our fMRI experiments so far can possibly capture the complex interaction between different brain regions while someone is considering and discussing whether he e.g. should leave a save work place or continuously give up his integrity.

I do not believe that we will never find out, I am just saying that departing from such a concept of free will, the experiment of Haynes’ group seems completely ridiculous. In other words, they observed something far from even having the potential to be a free will decision because free will needs something it is truly concerned with as well as time and interaction.

With this kind of imagery, it is not difficult to comprehend the power that dopamine wields over us when it is released, especially for those addicted to cocaine. Cocaine can actually inhibit dopamine reuptake via blockade of the dopamine transporter (DAT), increasing the synaptic availability of dopamine, prolonging its activity and producing the rewarding and addictive properties of the drug. DAT blockade by cocaine is correlated with the “high” experienced by addicts. The dopamine D2 receptor (D2R) plays some role in cocaine addiction, with lower levels of this receptor found in the nucleus accumbens (NAc) of cocaine addicts.

In a recent paper by Volkow and colleagues, the authors treated cocaine-addicted rats with D2R gene therapy in order to increase the levels of D2R in the nucleus accumbens (NAc). Up-regulation of D2R in the NAc was correlated with a decrease in lever pressing for i.v. infusions of cocaine, suggesting a possible therapeutic strategy in the treatment of addiction.

Methods

Rats were trained for 2 hours daily in operant chambers to press a lever for food pellets. After training, jugular catheters (for i.v. infusions) were implanted, along with guide cannulas leading directly into the NAc. After a recovery period of one week, the animals were re-introduced into the operant chambers. Now, rats received an i.v. infusion of cocaine following a lever press, with a 30 second time-out following each infusion. After 7 days of cocaine self-administration, rats then received a microinjection of an “empty” adenovirus, or a null vector, into the NAc. They were then returned to their chambers for another week of cocaine self-administration. Finally, all animals were given a microinjection of the D2R-expressing vector into the NAc and returned to the chambers for 14 more days.

Results

The number of infusions and lever presses were compared between (a) 1-7 days pre-null vector treatment, (b) 8-15 days post-null vector treatment and©16-22 days post-D2R vector treatment. After the animals were expressing increased levels of D2R in the NAc (because of the experimental vector), the average number of infusions decreased by 75% and the number of lever presses was also reduced.

D2R adenoviral infusion resulted in a decrease in cocaine self-administration and lever pressing for up to 6 days post-treatment, with this result eventually returning to baseline. A similar result was also observed for ethanol administration. However, the attenuated response of ethanol self-administration was of a longer duration than that observed for cocaine self-administration, suggesting that there are alternate pathways for dopamine reward. But at the very least, these results demonstrate the broad potential for such a strategy.

Discussion

In this study, D2R up-regulation disrupted cocaine self-administration, however the exact mechanism has yet to be elucidated. A possible explanation for these results is; cocaine use increases dopamine release, desensitizing or decreasing the levels of D2R in the brain and in effect rewiring the brain so that cocaine produces the feeling of reward. By increasing D2R in the NAc, there is an enhanced dopamine-D2R interaction, thereby increasing the sensitivity of the NAc neurons to the effects of the drug and lowering the dose of the drug that would be required to have a rewarding effect. With the animal receiving an equivalent reward with less cocaine, fewer lever presses (and subsequent infusions) would be necessary.

Questions

1. In the paper it is suggested that the D2R in the NAc is not the only route through which the pharmacological effects of cocaine take place; what are the other possible modes of action?

2. What other possible mechanisms may underlie how D2R up-regulation in the NAc decreases cocaine self-administration?

3. The attenuation of cocaine intake was transient in the virus-treated animals; what factors likely influenced this habituation to D2R up-regulation?

4. Is D2R gene therapy a viable treatment for addiction in humans?

These theories offer trade-offs in their ability to faithfully represent our surroundings—dense, global coding offers highly selective information storage and is resilient to damage, while independently meaningful neurons allow quick learning and preclude interference between codes. But recently, both theory and experiment have pointed to an optimal compromise between the two—sparse coding. In sparse coding, information is represented by a relatively small number of simultaneously active neurons, with the majority of neurons inactive.

The happy middle-ground of sparse coding allows efficient learning—there is low interference between codes, and changes can be governed by local, e.g., Hebbian, rules—high memory capacity, simple transformations, and a substantial amount of information storage. Sparse coding is now known to be widespread in the nervous systems of many animals—including the rodent visual, motor, barrel, and olfactory systems, the zebra finch auditory system, and the cat lateral geniculate nucleus—but the sparseness of representations in the rodent auditory cortex has not been explicitly addressed.

Which brings us to this week’s paper—Sparse Representation of Sounds in the Unanaesthetized Auditory Cortex —by Tomáš Hromádka from Tony Zador’s lab at Cold Spring Harbor, which came out in PLoS Biology. To investigate the sparseness of auditory coding, Hromádka surveyed how the neuronal population of the primary auditory cortex responded to particular stimuli.

Unable to analyze every neuron in the auditory cortex, Hromádka optimized his technique to get as unbiased a survey as possible. First, to isolate a representative sample of cells, he used cell-attached recordings with glass electrodes—allowing him to find cells by detecting resistance, rather than poking around looking for flurries of big spikes (which tends to bias other recording techniques toward highly-active neurons).

Second, to determine the population’s response as a whole, they presented each neuron with identical sound repertoires (tones, sweeps, white-noise bursts, and natural sounds), rather than optimizing the sounds for each neuron’s responsiveness.

And third, the animals were awake. Bartiturates and ketamine, which are generally used in global anesthesia, alter neuronal response properties, leading to transient, homogeneous responses. But by using awake animals (with their heads in a stereotaxic rig and their bodies in a plastic tube), they could get the full, natural diversity of neuronal responses.

So they presented their tones, listened for the auditory code, and heard—radio silence.

Only half of the neurons tested showed any change in firing rate for any stimulus presented, and less than 5% of the entire population showed a “well-driven response” (which they defined as a change of at least 20 spikes per second). Further, both the evoked and spontaneous firing rates were low—and in both cases, most of the spikes were actually generated by small subsets of neurons.

In the evoked condition, most of the neurons ignored the stimuli, while a few neurons fired vigorously in response to certain frequencies. In the spontaneous dataset, the authors suspect that the small subset of active neurons may have been broadly-tuned interneurons (although they didn’t actually identify cell-types)—which likely contributed to the overall silence of the population.

Of course, information about how individual neurons behave is somewhat useless on its own. The interesting issue is the code that represents the sounds to higher areas and ultimately guides behavior. This code must take into account not only the highly active neurons, but also any neurons that respond at all. When the authors analyzed such population-level responses to the tones and the sweeps, they found that, at any one time, only about 10% of neurons showed any significant increase in firing rate, and even fewer showed a significant decrease.

These results suggest, for the first time (according to the authors), that sounds are represented in the auditory cortex as sparse codes, strengthening the argument that sparse coding may be the guiding principle for cortical representations of sensory input.

Discussion Questions

1. How would attention/emotion affect the sparseness or other properties of the code?
2. How might sparse coding contribute to the cocktail party effect ?
3. Sparse coding is often proposed as a means to conserve energy—we might not have enough resources for any other type of coding. If this is the primary driver of the evolution of sparse coding, it’s possible that our coding system is sub-optimal, constrained by our anatomy and energy requirements. Which—metabolic advantage or representational power—would you argue is the greater evolutionary force?
4. The more selective a code, the larger the number of neurons required. It has been suggested that invariance allows highly sparse codes to be selective. What mechanisms might underlie this invariance?

Now, tragically, I actually live in Illinois. When I tell Illinois people that skunk story, they don’t laugh very much. Once in a while, they even ask, “How do you know those guys were from Illinois?” To which I respond: “If they weren’t from Illinois, they wouldn’t have acted like that.” Then I give them the same look I give people who say they don’t like demolition derbies, and punctuate it by reaching into the cooler for another Leinie’s. Once, after telling the skunk story to an Illinois friend, I reached into the cooler and there weren’t any beers left. I was swishing my hand around in the water to make it clear we needed more supplies, but the guy I was drinking with just sat there looking at me unsatisfied. Normally, I wouldn’t spend too much time on the subject with people who obviously don’t know the field, but my mouth was getting pretty dry since finishing the last beer and it was still well before noon, so I decided to go all intellectual: “Well, over decades of study, we Wisconites have identified a number of markers that can be used to reliably distinguish people from Illinois.” He kept sitting there, expectantly, so I continued. “For example, Illinois people drive really fast through town even when they’re sober. And they don’t wear brown corduroy suits for church and holidays like normal people do. Neither of those skunk-catchin’ boys had a brown suit, and they drove pretty fast. That’s two pieces of evidence, right there, in addition to the whole skunk thing.” “Besides,” I added, “me and my family have been telling that story for years, and since the beginning those Illinois boys have always been identified as Illinois boys.” I leaned back and burped, to emphasize the fact that differentiation between Illinois and non-Illinois people was a highly developed science and any questioning of my conclusions was a sign of ignorance. After he had refilled the cooler and we had cracked another couple cans, I even told him the rest of the story. “The funny part is, that car that those Illinois boys stunk up wasn’t even their own. It had Wisconsin plates!”

Questions for discussion:

1) Were those really Wisconsin boys who just happened to drive fast and not wear brown corduroy, meaning that driving fast and lack of brown corduroy are not really reliable markers for Illinois people?

2) Beyond lineage analysis (e.g. Wisconsin people are from Wisconsin, and Illinois people are from Illinois), are there really any reliable categorical differences between Wisconsin and Illinois people?

3) Is the distinction between Illinois and Wisconsin people really useful, or is it an arbitrary distinction, historically rooted?

After you’ve thought about your answers a bit, read the recent paper by Karadottir et al published in this month’s issue of Nature Neuroscience. This paper shows that there are (at least) two types of oligodendrocyte precursor cells in rat P7 cerebellar slices. All OPCs were identified as OPC glia based on the presence of the traditionally ‘OPC glia specific’ markers NG2 & OLIG2, and the absence of a traditionally ‘neuronal-specific’ marker, NeuN. But about half of all OPCs examined had large voltage-gated sodium and potassium currents, fired action potentials, and had glutamatergic and GABAergic synaptic inputs. In other words: There were no obvious electrophysiological differences between neurons and many OPC glia. Depending on your point of view, this news could be either very exciting or deeply disturbing. Either way, the study and the fact that it is published in Nature Neuroscience reveals as much about neuroscientific culture as it does brain function.

Were the ‘OPCs’ studied by Karadottir really neurons that happen to express some ‘glial’ markers? How does one distinguish neurons and glia anyway without relying on markers? If there is no reliable way to categorize a cell as ‘glial’ or ‘neuronal’ without the use of markers, then how can we assign markers as ‘glial’ or ‘neuronal’? Is the continuing functional distinction between ‘glial’ and ‘neuronal’ cell lineages an ultimately disastrous conceptual mistake? If the nervous system is not just neurons (we all know it’s not), and neurons aren’t the only fast electrical signaling, synaptic contact-making cells carrying important information in the nervous system (many studies over the past couple decades have shown that they’re not), then why do we keep calling our field ‘Neuroscience’? Should we simply go back to being ‘physiologists’ or ‘cell biologists’ or ‘behavioral scientists’?

References:

Karadottir, Hamilton, Bakiri, and Attwell, “Spiking and nonspiking classes of oligodendrocyte precursor glia in CNS white matter” Nature Neuroscience 11(4):450-456 [April 2008].

The experimental setup was beguilingly simple (by today’s standards): show a visually fixating monkey differentially oriented sine-wave gratings while recording from cells in primary visual cortex (V1). It is known that cortical cells will rapidly adapt after being exposed to redundant information for several hundred milliseconds. Thus, the authors recorded responses before and after a 400-ms adaptation to a sine-wave grating of fixed orientation. The test stimulus shown to the monkey after adaptation was a movie of sine-wave gratings presented at 60 Hz.

A pair of cells preferring nearby orientations (less than 30° apart) or very different orientations (more than 60°) exhibited a strong reduction in their correlated activity. In addition—and somewhat confusingly—cell pairs that preferred nearby orientations showed significant decorrelations only when the difference between the preferred orientation and the adapting stimulus was again small or large, i.e. less than 30 or more than 60 degrees. Finally, not only does adaptation reduce the strength of correlations, but it also reduces the variability of correlation.

The authors hypothesized that changes in coding elicited by adaptation would increase the amount of information stored in the network of cells. They estimated the information held in the population from the mean firing rate and covariance matrix of the individual units. This metric, the Fisher information, provides an upper bound that any putative neural decoder could use to extract the stimulus orientation. If changes in the mean and variability of the correlations are taken into account, the post-adaptation discrimination threshold is improved by 40%. Test stimuli that are similar or very different from the adapting stimulus provoked the largest improvement in coding efficiency. Finally, the authors point out that yielding a similar improvement by adjusting firing rate alone would have required an increase of 55%, certainly high enough to be metabolically costly.

This paper highlights one mechanism by which a system’s input-output curve can be modified on the fly. Other evidence along these lines has been accruing for years. Work by Reynolds et al. showed how attention can increase the sensitivity of V4 neurons. Even human psychophysical data, mentioned in the paper, are in agreement with this mechanism. In David McCormick’s lab, work has shown how network activity can influence individual neuron’s responsiveness, ultimately modifying how stimuli are represented (Hasenstaub et al.) Recent work highlighted by Cori Bargmann at the Neuronal Circuits meeting showed multiple ways in which the activity of identified circuits in C. elegans could be modified by the environment. She pointed out that even with knowledge of all the connections in this model organism, we may still understand less than half of how that system functions. These few examples underscore the importance of understanding the dynamics of a system, as Gutnisky and Dragoi have begun to do here.

Discussion Questions:
1. The authors mention that this mechanism is a “metabolically inexpensive solution.” What other advantages does it have?
2. The authors point out one downside of this mechanism is that it could interfere with other information processing. Are there other downsides to decorrelating inputs?
3. Is the proposal that this mechanism may optimize image-discrimination performance in real time feasible? Saccades may take anywhere from 20 to 200 ms, and occur many times per second. 400-ms adaptations are performed here. How many times can this adaptation occur in sequence?
4. As a rough generalization, one could think of this paper as highlighting the importance of circuit dynamics over circuit connectivity. Though the interconnections of the circuits here are not known, much can still be gleaned from how the system stores information (which after all, could be considered the ultimate goal in this instance.) If all-to-all connectivity is true, or if the specificity of connections is not high, then indeed it is not connectivity that matters but in fact dynamics. What impact will this have on the current era of ‘connectomics’?

Mapping the functional connectivity of the brain is both a very old and currently rather hot topic in neuroscience. Remarkably, using the simple technique of the Golgi stain, Ramon y Cajal was able to describe the morphology of numerous cell classes in the brain and also infer the functional anatomy of circuits such as the hippocampus. He was helped by the highly stereotyped circuit architecture of some brain areas. Despite a century of follow-up work, our knowledge of the overall wiring diagram of the brain remains at a rudimentary stage. Modern neuroscientists need a reliable and simple method to identify the connections between genetically selected cell types in complex local circuits and from long distance connections.

A recent paper by Feinberg et al, from Cori Bargmann’s lab, should help us make sense of these tangled webs of connections. The authors propose a clever new technique, Genetic Reconstitution Across Synaptic Partners, to track the locations of selected synaptic connections backed by an impressive set of in vivo proof-of-principal experiments in c. elegans. The gist of the strategy is to split a fluorescent marker into two non-functional components and then distribute each half on different sides of circuit’s connection. Only at synaptic connections would the two components be close enough to undergo trans-complementation and reconstitute a functional marker.

A number of labs had previously shown that the green fluorescent protein (GFP) could be split into two non-fluorescent halves, which can trans-complement and become fluorescent. However, these probes generally had poor kinetics and required tethering by fusion to tightly dimerizing accessory components to prevent probe degradation. These requirements presented too much of a challenge for trans-synaptic complementation. Thankfully, in 2005 the Waldo group published their ‘superfolder’ GFP, which also turned out to be more much stable when split to two components. Results
Feinberg et al. took this split superfolder GFP and genetically expressed each half on the surface of neurons. They used a variety of tethering strategies, including fusion to the broadly distributed transmembrane protein CD4, the presynaptically targeted PTP-3A and the pre and postsynaptically targeted (in the worm) neuroligin homolog (NLG-1). When expressed in worm lines, each technique was able to generate stable fluorescent labels at the sites of synaptic contact between genetically selected sells. With fluorescence microscopy, they used the probe to visualize sites of known synaptic contact and to discover novel synaptic mistargeting in several mutant lines.

GRASP shows great promise to define the connectivity map of the brains of drosophila, zebrafish and mammals. This technique could find significant use in studying the patterns of experience dependent rewiring of synaptic connections in small transparent in vivo systems. Similar work in mammalian systems would likely be restricted by light scattering, but single timepoint measurements could be taken across the entire brain with careful tissue fixation. The relatively small genetic size of the probes should facilitate methods of viral introduction, making transgenic line generation unnecessary for many questions. Speaking of questions, this paper makes a few discussion questions come to mind…

Questions:

1. In some of the images there are high levels of autofluorescence that are brighter than the labeled synapses. What is the source of this background? Would it be as much of a problem in mammalian brains? 2. How would you confer greater synapse specificity in mammalian systems? What effects would you have to be watchful for if you overexpressed synaptic adhesion molecules in a mouse? How could you alleviate these problems? 3. Interestingly, the spilt GFP site is very asymmetric, with a 16 residue peptide (215-230) complementing a 214 residue GFP barrel. What impact would this have on extending the technique to include multiple fluorescent colors, such as CFP and Citrine What about for the mFruits? 4. This technique labels the close juxtaposition of two neurons. In what ways could you prove the label was targeting functional synaptic connections? How could you determine the polarity (excitatory vs. inhibitory) of the connection? 5. Would it be possible to reconstitute functional activity sensors at synapses? For example could this strategy be used to reconstitute GluSnFR and image glutamate at a specific neuronal connections? What additional challenges might this entail? 6. What advantages does GRASP have over Steve Smith’s Array Tomography, or Jeff Lichtman’s Brainbow technique for mapping synaptic connections? What are the disadvantages?

The human body senses external inputs and responds to these conveyed signals through a complex neuronal network. Sometimes, to probe the complexity, neuroscientists have to think small and focus on the working unit of synaptic transmission, the synapse. Synapses consist of both a presynaptic and postsynaptic compartments, and it is essential for these two compartments to be properly aligned for accurate assembly. Several molecules that mediate trans-synaptic cell adhesion and signaling have been suggested to be essential in maintaining the integrity and the function of synapses. A paper published last year in Neuron by Li et al. described the crucial role of one such molecule, Neurexin, during synaptic transmission in an intact organism, Drosophila melanogaster (available here).

Neurexins are receptors for alpha–latrotoxin, a neurotoxin that triggers massive neurotransmitter release. Unlike mammals, which express three neurexins, there is only one neuroexin gene in Drosophila (Drosophila neurexin, dnrx). Dnrx is expressed in central neurons and is localized in the active zones, as suggested by work conducted in the Drosophila neuromuscular junction (NMJ). Genetic studies on dnrx showed that flies lacking dnrx exhibited abnormal NMJ structures and reduced synaptic bouton numbers, suggesting that dnrx is involved in NMJ expansion and synapse formation. Moreover, elevated dnrx expression in neurons, but not in muscle, increased synaptic bouton numbers, suggesting that dnrx expression is required for the proliferation of synaptic boutons in the pre-synaptic compartment.

How does dnrx regulate synapse formation and development in vivo? Experimental results provided by Li et al. have nicely addressed this question. First, the authors found that for dnrx mutant synapses, both the distribution of pre-/post-synaptic proteins and the active zone structure were altered. The number of T bars (part of the NMJ active zone structure) per bouton was increased, and both the presynaptic density (PRD) and the postsynaptic density (PSD) were over 60% longer. Defects in mutant dnrx synaptic ultrastructure correlated with synaptic transmission defects, as monitored using electrophysiology. These results suggested that dnrx regulates synaptic formation and development for proper synaptic transmission.

Similar to observations in mammals, flies lacking dnrx abnormally sensed calcium levels, but the distribution of presynaptic calcium channels was unchanged. This suggested that neurexins regulate the coupling between calcium channels and neurotransmitter release machinery. Most strikingly, although the PRD and PSD of dnrx mutant synapses were properly aligned, the PRD exhibited signs of detachment from the PSD at several points. A lack of dnrx had resulted in an adhesion defect between the pre- and postsynaptic cells, causing disruptions in normal synapse formation and function.

Thus, this study has established an in vivo role for neurexin in defining synaptic architecture and providing for proper synaptic transmission. Future work on identifying and characterizing Drosophila neurexin partners will help to elucidate the in vivo function of neurexin.

Discussion Questions:

3) What experiments could have strengthened the evidence suggesting the potential functional interactions between neurexin, Ca2+ channels and neurotransmitter release?

In a recent paper (available here), Marco Contreras, Francisco Ceric, and Fernando Torrealba examined the role of insular cortex in drug craving and affective states. Amphetamine-experienced rats were trained to exhibit conditioned place preference for an amphetamine-associated compartment. Conditioned place preference illustrates how a drug-induced subjective experience can become associated with environmental cues. A brief description of the methodology: animals are given injections of a drug, in this case amphetamine, and placed in a chamber with distinct contextual cues. Other animals are given saline and placed in neutral chambers. After several injections, the animals are then placed in a third compartment, one with clear access to either of the first two chambers, and allowed to choose where to spend their time. If a drug produced a subjective state that is rewarding, animals will spend more time in the drug-associated chamber. This method provides an indirect measure of reinforcement and allows researchers to determine how well animals can differentiate the internal states that different drugs create.

In the current study, amphetamine-treated animals preferred the amphetamine chamber, as expected. Researchers then reversibly inactivated their insular cortices with lidocaine, a sodium channel blocker. Inactivated animals reversed place preference to the non-amphetamine-associated chamber, exhibiting preference similar to saline controls (controls preferred the non-amphetamine-associated chamber because it was darker). This effect reversed again after the lidocaine wore off, with the amphetamine-experienced rats returning to the amphetamine chamber. Lidocaine injections into the adjacent somatosensory cortex did not disrupt conditioned preference for the amphetamine chamber. The researchers conclude that insular inactivation reduced drug craving; rats spent less time in the amphetamine chamber because they no longer felt the subjective state that made amphetamine rewarding.

To examine insular involvement in the development of aversive internal states, researchers induced a “malaise-like” state with lithium chloride (LiCl). Inactivation of the insular cortex blocked the malaise-inducing behavioral effects of LiCl. In rats, this state is characterized by a sagging posture and decreased locomotion. Lithium chloride is often used to treat the mania aspect of human bipolar disorder.

Specificity of insular activation was tested with Fos immunoreactivity. Fos is the protein product of the immediate early gene c-Fos often used as a marker of neuronal activity. Conditioned place preference to amphetamine and LiCl administration significantly increased Fos protein expression in the insular cortex relative to controls. Utilization of axonal tracers verified that lidocaine injections were effective at inactivating the primary insular cortex, which includes the posterior granular insula and projections to visceral-thalamic, limbic, and prefrontal regions.

If the insula is important for generating motivation to take drugs, its inactivation should decrease drug craving, as the researchers note. The authors cite a recent human finding showing that subjects with insular damage were able to easily quit smoking. This relationship between drug craving and insular activity has also been backed up by neuroimaging studies. The insular cortex is active when addicts are exposed to environmental cues that signal craving. Interestingly, humans with insular damage do not report decreased food craving or intake. The authors suggest this implicates the insula as part of a system for measuring state of well being. Strong deviations from homeostasis, like those elicited by drugs, activate the insula in part to mediate motivation and aversion.

Given these findings, it is clear that the involvement of the insular cortex in drug craving and affective states is important for the study of addiction. Most drugs of abuse act on natural homeostatic and reward pathways, changing them in ways that promote compulsive drug craving and seeking. In humans, such changes can persist for years, making relapse a perpetual danger. Future research will need to examine how the insula, through interactions with mesolimbic dopamine pathways, prefrontal cortex, and memory systems, acts to develop and maintain subjective drug experience.

Discussion Questions:

5. The idea that the insula is a “well-being detector” has interesting evolutionary implications. Why would this system have evolved to be so susceptible to compounds of abuse, which initially provide some signal of well being, but later cause such deleterious consequences?

Briefly, a paper published by Linda Buck in 2001 was recently retracted in Nature. Now, a retraction always turns a few heads, especially when it involves a high-profile publication, and ESPECIALLY when it involves the work of a Nobel prize-winning scientist. But what makes this particular retraction even more interesting is the fact that an “Author Contributions” abstract was included by the authors with the retraction. It makes it pretty clear that the other authors place most of the blame for the irreproducibility on the first author.

Now, obviously, The Buck lab is doing the right thing by retracting a paper containing results that they can no longer reproduce, but it seems a bit odd to isolate one author when there was actually a “co-first authorship” on the publication. So the other first author should get the glory, but be protected from the retraction wrath? What do you think? Discussion topics about retractions in general:

1. For a retraction, should dissenting authors be given the opportunity to respond to the allegations against them before the retraction is published?

2. Continuing from the previous point, in that case, should the results and “case” of each side be presented for peer review before deciding on the retraction?

3. If your name is on a fraudulent manuscript, are you as equally liable as the perpetrator in the retraction?

4. It is obviously not practical for a productive lab head to review every single data point for every single figure in every single publication…or is it? In other words, how much trust should a PI afford to his/her students and post-docs?

The study of visual consciousness in cognitive neuroscience is based on establishing correlations between three parameters: properties of visual stimuli, brain activity and the conscious state of the subject (communicated by means of verbal or non-verbal behavioral reports, also called “psychophysics”). Progress in this line of research is often defined by how well the researchers control the timing of stimulus presentation, how accurately brain activity is measured and whether the behavioral reports are precise.
The paper by Del Cul, Baillet and Dehaene (2007; free PDF here ) introduces a new methodology to sharpen the control of visual stimuli and examine the respective effects on brain function. After a previous study on subliminar (unconscious) perception elicited by brief stimulation (ref. 21 in the paper), Stanislas Dehaene and colleagues measured brain events and behavioral reports elicited by the presentation of supraliminar stimuli combined with backward masking. The main goal was to find the sequence of brain events necessary for the formation of a reportable conscious visual state.
The authors attempt to address a controversy in their field concerning which brain areas are essential for human visual consciousness. Researchers in one camp claim that the activation of visual areas in occipital cortex is sufficient to generate visual conscious states. A second group suggests that the activation of other cortical associative areas (especially frontal visual areas) is also necessary. The timing of brain events supporting conscious processes is crucial to distinguish between the diverging views, since processing in occipital areas alone would take around 100-200 ms to occur, while the formation of a large-scale integrative process involving more brain areas (frequently related to the Global Workspace Theory, proposed by Bernard Baars in 1989) would take longer.

Methods and Results

The target stimulus consisted of a single digit (a number) projected for 16 ms (a brief, but supraliminar stimulus). The mask was a group of numbers projected soon after, for 250 ms, at the same visual location. The authors varied the time interval (called SOA, “target-mask stimulus onset asynchrony”) between stimulus onset and the presentation of the mask, from 16 to 100 ms. Shorter intervals were predicted to cause backward masking of the stimulus (basically, relegating the stimulus below the threshold of consciousness), by means of a perturbation of the sequence of brain events necessary to generate the corresponding conscious state. Longer intervals would not perturb the brain processing of the stimulus, allowing the propagation of excitation to higher cortical areas.
High-density event-related potential (ERP) recordings were collected to determine whether a change (or a “transition”) in brain activity occured between the lower and higher SOA values. The temporal location of this transition was defined behaviorally, using two measures: a forced-choice comparison of the presented digit with another one (checking for both sub- and supraliminar perception), and a scale of visibility, which roughly assesses the conscious access the subject had to the stimulus.
This methodology produced several behavioral and physiological (ERP) results. Behaviorally, a “significant nonlinearity” was found for the SOA interval from 33 to 66 ms. Below 16 ms, the performance was at chance level, suggesting that the presented digit was completely masked by the second. In the 16-33 ms interval, performance in the forced-choice task was above chance, while in the 33-66 ms interval both the forced-choice and the (conscious) visibility ratings increased non-linearly. Above 66 ms there was not a significant change in visibility and the subjects consistently had conscious access to the presented stimulus. The authors concluded that “a major transition in processing occurs around SOA = 50 ms”.
The authors then looked for ERP components temporally correlated with the transition in visibility elicited by the 50 ms SOA. Using statistical testing across subjects, they found activity within a fronto-parieto-temporal network (called the P3 component in the paper) to be strongly correlated with the non-linear increase in visibility. The change in P3 that correlates with the transition occurs about 270 to 300 ms after target onset. This finding is consistent with the hypothesis that conscious access to a visual stimulus involves the sequential activation of several cortical areas. Thus, we now have some fairly strong evidence suggesting that the conscious process of visual stimulus sensation involves more than the simple activation of occipital visual areas. So, in order to cross the threshold to consciousness, we need network activity that is likely greater than the sum of its parts.

Questions:

1) Why is activation of frontal visual areas necessary for (conscious) visibility?

2) What are the physiological mechanisms supporting the Global Workspace?

3) Does conscious processing in other perceptual modalities also display similar timing?

Lidocaine’s lack of selectivity is hardly surprising given the drug’s mechanism of action: it works by blocking voltage-gated sodium (NaV) channels, which are of course vital for the propagation of action potentials. In a paper recently published in Nature, Binshtok and co-workers describe a rather novel and clever strategy for selectively inhibiting only the NaV channels expressed on nociceptive neurons. To do this, they used a quaternary derivative of Lidocaine, QX-314, which is usually ineffective as a local anesthetic because it is not membrane permeable. Lidocaine-related anesthetics can only block NaV channels from the inside of the cell, and so must readily cross the plasma membrane in order to be active. QX-314 cannot enter the cell, and thus cannot inhibit NaV. However, another ion channel chiefly expressed in small diameter nociceptive neurons, TRPV1, is known to have a particularly wide channel pore capable of transporting relatively large molecules across the membrane, including organic molecules such as N-methyl-D-glucamine (NMDG) and the dye FM1-43. The authors hypothesized that QX-314 might be able to enter the neuron through activated TRPV1 channels, and subsequently block NaV channels and pain transmission. The idea being that this way only pain-transmitting nociceptors would be blocked by QX-314.

Anyone with a penchant for spicy food will be familiar with the function of TRPV1 receptors, which confer our sensitivity to the chili pepper ingredient, capsaicin. TRPV1 receptors are polymodal cation-permeable channels that open in response to a diverse array of noxious stimuli, including high temperature, acid, ethanol, spider venom toxins among others.

Using voltage clamp electrophysiology, the authors measured the amplitude of NaV currents evoked by depolarization of the cell membrane in isolated rat dorsal root ganglion (DRG) neurons. As expected, application of QX-314 alone to the bath solution had no effect on NaV currents evoked in DRGs. However, when capsaicin (used to activate and open the TRPV1 channels) and QX-314 were co-applied, the amplitude of the NaV currents were significantly reduced, while the NaV currents of large diameter, capsaicin-insensitive neurons were unaffected. The authors then carried out current clamp experiments where they showed that co-application of capsaicin and QX-314 significantly disrupted the initiation of action potentials in nociceptive neurons, consistent with the effect of inhibiting NaV channels.

Turning their attention to the whole animal, the authors tested whether the co-administration of capsaicin and QX-314 could selectively block pain transmission in vivo. They injected capsaicin and QX-314, separately or together, into the rat hindpaw, and tested the effects of these drugs on two withdrawal models of pain. First, they looked at how much force could be applied to the paw with a microfilament (von Frey filament) before the rat withdrew it. They discovered that 1 hour after co-administration of QX-314 and capsaicin, there was a significant and long-lasting increase in the amount of force that the rat could tolerate before withdrawing its paw. Next, the authors tested the effect of drug administration on the latency of withdrawal in response to a noxious heat stimulus to the hindpaw. Again, 1 hour after co-administration of capsaicin and QX-314 there was a significant and persistent increase in the latency of paw withdrawal, suggesting that the animal could tolerate higher temperatures before feeling discomfort. When applied alone, QX-314 had no effect on either of these responses. Capsaicin on the other hand actually decreased the rat’s tolerance for both mechanical and thermal stimuli, consistent with sensitizing nociception.

The next question to address was whether motor neurons were affected by the co-administration of capsaicin and QX-314. To test this, the drugs were injected separately or together in close proximity to the sciatic nerve, the main nerve branch extending into the leg. Consistent with the previous set of experiments, the presence of both QX-314 and capsaicin significantly increased the subject rat’s tolerance to both mechanical and thermal pain stimuli. However, the presence of these drugs had only a negligible affect on locomotion, showing that motor function was not substantially impaired. In contrast, application of Lidocaine severely disrupted locomotion in addition to suppressing pain transmission, as would be expected for a drug that effectively blocks all neuronal activity.

In summary, the authors of this paper succeeded in selectively delivering anesthetic to pain transmitting neurons, and in doing so significantly reduced the side effects usually associated with NaV-blocking drugs.

Discussion Questions:

Q1. Is this procedure therapeutically viable?
The time taken to establish a therapeutic effect is arguably only a minor drawback. The co-administration of QX-314 and capsaicin does admittedly take in excess of 30 min to be effective. In contrast, Lidocaine is usually maximally effective within a shorter time period of 5 to 15 minutes. However, an extra 20 minutes or so might be a small price to pay to avoid the indignities of the drooling, tongue lolling, rubber-faced side effects.

Q2. Could a similar procedure be used to attain specificity with regard to the application of other types of drugs to other types of cell?
The significance of these data arguably extends beyond the use of local anesthetics. TRPV1 receptors are not the only ion channels that are known to conduct large molecules across the membrane. Certain members of the adenosine 5’-triphosphate (ATP)-sensitive P2X receptor channels have been known to do so as well. Therefore, it is certainly plausible that the strategy described by Binshtok and co-workers could be applied in the delivery of other intracellularly-acting drugs to different cell types.

Question 1: Do all the examples I have just given call on similar neural pathways?

Question 2 (the BIG one): I remember someone saying on the radio “The thing that most shocks me is that I am not shockable anymore!” She was talking about how she couldn’t get shocked any more, looking at the news. It was this fact that shocked her the most!

In this day and age, we can see stories of tragedy unfold in the four corners of the globe simultaneously.

The question is: what impact does this torrent of negativity have on the neural pathways mentioned above and do you think this is what was desensitizing the woman? The bigger question is, are the media inadvertently contributing to a world where we are becoming increasingly polarized in our feelings?

On the one side: Mirror neurons and pathways related to empathy and pain are triggered when we see a child who has lost both her parents in civil war. But on the other side, it doesn’t shock us so much any more and we are less likely, as a society to take action. Our threshold at which we attend to a given problem has increased.

What are the moral implications of this and do scientist (specifically neuroscientists) have a role to play in the solution? (assuming there is any truth in what I have said).

Maybe this issue has been addressed in which case, I would be very grateful for some references. Am I right to think that is a massive problem for society and us as a species?

You know when your driving (or walking or cycling) to work and when you arrive you can’t remember your journey, because you’ve done it so many times?

Question 1: Which part of the brain is responsible for putting you on autopilot and what evolutionary benefit does it carry? I imagine it has something to do with reducing energy consuming (why should it be more energy consuming anyway) top-down activity, but I need an expert explanation(s).

Question 2: The mechanism which kicks in when an anomaly is detected in your journey (e.g. an old lady steps out in front of your car) waking you up from your day dream. What exactly is happening here? I have found one reference which only tentatively explains this in terms of a threshold being crossed in the ‘ambient scanner’ triggering top-down (concious) neural activity and an involuntary reorientation of attention. But the reference is 17 years old. What is the latest?

In your answer, I would prefer some references as I like to read about this stuff. I am particularly fond of the cool names that you have for different areas of the brain like ‘superior frontal gyrus’. So please pile in the names in your answer!

I have another burning question related to my first question that I am posting in a different topic, because I think it should be discussed separately. If possible please take some time to read it.

Thanks! Haydon

Why start with birds? Unlike mammals, birds can regenerate hair cells throughout their lives and even following multiple rounds of deafening. Furthermore, for unknown reasons, vestibular tissue (in both birds and mammals) is more likely to spontaneously regenerate hair cells than cochlear epithelium. So, embryonic avian vestibular tissue was a logical starting place for the development of an in vitro hair cell differentiation model. Their methods were relatively simple: dissect away an interior portion of the E14 chick utricle, dissociate the cells in 6 or 7 passages, start a new cell culture in suspension, and then observe the morphology of the resultant spheres.

Following the dissociation of the vestibular epithelium, the hair cells and supporting cells began to lose their epithelial junctions, with a concomitant reduction in E- and N- cadherin expression (proteins important for making cell junctions). They transitioned into a mesenchymal phenotype, and began to grow as solitary cells rather than as epithelia. After that transition, cells could be dissociated and allowed to thrive in a suspension, which prevented them from adhering to substrates as opposed to each other. Within a few days, the cells began to aggregate and form hollow spheres. As early as day six after suspension, some of the cell spheres started differentiating into hair cells. This was concluded based on the expression of the hair cell markers myosin VIIa, calretinin, parvalbumin 3, and otoferlin. Two days after this initial expression, actin-filled stereocilia could be seen projecting from 3-5% of cells in the sphere (the same cells which were positive for hair cell markers). Four days later, about 15-20% of the sphere’s cells had become hair cells. It is interesting to note that the hair cells’ stereocilia always projected away from the sphere’s lumen, into in the surrounding media. Other cells comprising the sphere were positive for the supporting cell marker SCA, suggesting that the spheres were capable of producing diverse cell types mimicking the auditory epithelium.

However, it is important to prove that these new hair cells are functionally, as well as morphologically, well-developed. This can be tested with a dye called FM1-43, which enters and stains an active hair cell through open mechanosensory transduction channels. When Corwin’s group incubated spheres (passaged 15 or 19 times) with FM1-43 for 10 seconds, only the hair cells in the spheres were labeled with the dye. This suggests that the resultant hair cells from this group’s cell culture method have developed functional mechanosensory transduction channels.

In addition to providing an in vitro population of hair cells which could be useful for drug testing or studies on development, this work has important implications for the replacement of human hair cells. Corwin’s group was able to generate new hair cells from a homogenous line of cells which had been frozen, thawed, and exponentially expanded. The cells survived for months in culture, and did not require implantation into the inner ear before differentiating into functional hair cells. The next step might be to implant these new hair cells into vestibular or auditory avian epithelium in order to observe whether they are capable of integrating into the cellular matrix, and ultimately, within the auditory or vestibular circuit.

1. What might prevent the same culture methods from being used with human hair cells?
2. Are there explanations for why FM1-43 entered hair cells, other than through functional mechanosensory transduction channels? How might this be tested?
3. Why might birds and reptiles have retained the ability the regenerate hair cells?
4. Why might mammalian vestibular hair cells retain the ability to regenerate while cochlear hair cells have not?
5. Integrating new hair cells into the mature epithelium might prove to be daunting in humans. What might improve the feasibility of this approach?

In layer IV of S1, each whisker is represented by a ‘barrel’, a 500 micron-wide circular region of cortex. The stereotyped and relatively simple arrangement of the whiskers and barrels have made the whisker system a popular and fruitful model system for studying the cortical processing of sensory information.

Methods and results
Spatiotemporal Dynamics of Cortical Sensorimotor Integration in Behaving Mice, by Ferezou et al. (paper available here), is an experimental tour de force. They applied a voltage-sensitive dye (VSD) to the cortex of head-fixed mice to measure subthreshold voltage changes in the cortical surface (layers II/III). They analyzed the activity in a large area of cortex that included S1 and primary motor cortex (M1). This allowed the authors to examine sensory responses in S1, M1, and the relationship between responses in these two areas.

In the first set of experiments, they stimulated a single whisker in anesthetized mice. The earliest cortical response was localized to the corresponding barrel in S1. Surprisingly, within 8 ms this localized response spread to the entire whisker representation in S1 as well as M1. Interestingly, they found that different regions of M1 were activated by different whiskers: that is, there is a map of the whiskers in M1. In addition, S1 activation and inactivation experiments showed that that S1 activity was both necessary and sufficient for stimulus-dependent responses in M1.

The second set of experiments was performed in awake, head-fixed mice. When a mouse moved a whisker into an obstacle, the same pattern of cortical activation as described above was observed (i.e., localized S1 activation followed by broad S1 activity and M1 activity). Indeed, Figure 7B shows a trial in which most of the cortical hemisphere was activated within 40 ms of the touch.

Finally, the authors demonstrated that the sensory response is strongly influenced by the general behavior of the mouse during whisker stimulation. For example, when the mouse was initially resting but started to whisk in response to whisker stimulation, the same pattern of activation described above was seen. However, different responses were observed when the stimulation did not evoke whisking behavior in resting mice or when the stimulus was delivered in the middle of a whisk cycle.

Discussion
Ferezou et al. showed that subthreshold responses to whisker stimulation can be quite broadly distributed, often extending into M1. This suggests that M1 does not have a purely motor function, but serves also to process sensory information. While M1 projects directly to the brain stem and spinal cord to coordinate motor activity, its tight link with S1 opens up interesting questions about its role in sensory processing and sensorimotor transformations. Also, the sensory response in S1 and M1 depends on the behavioral state of the animal, suggesting that sensory processing isn’t a stationary process, but is sensitive to the context in which a stimulus is delivered. So, when someone asks how a mouse’s cortex would respond to a given stimulus, you probably have to ask, “What is the critter doing?”

Discussion questions
Note: some of these are mentioned in the paper.

1. How broadly does the sensory response spread? Does it spread to visual, auditory, and other sensory cortices (and vice versa)? What implications would such broad sensory effects have for theories of sensory processing in more ethologically realistic contexts when inputs are coming in from multiple modalities?

2. In nature, mice explore objects with many whiskers simultaneously. Would you expect the results in the paper to generalize to more naturalistic stimuli?

3. What are some possible functions of the observed differences in the sensory response when the mouse is in different behavioral states?

4. When the awake mouse was whisking and contacted an object, the typical S1->M1 pattern was observed, but when the whisker was stimulated magnetically during a bout of whisking (the final set of experiments mentioned above), the response looked quite different. What might explain this seeming contradiction in their results?

5. Rodents can still whisk when their cortex is removed (there seems to be a brain stem CPG responsible for whisker movement). What types of control does M1 exert over whisker movements in intact awake rodents? What role does S1 play in such motor control?

6. Rodents have fewer somatosensory areas than primates. Would we see the same sensory influences in M1 of primates?

Although interesting, there are plenty of problems with this study, the most glaring being that participants were randomly selected as opposed to using individuals with established paranormal talents. But independent of that, and despite the nice design of the experiment, the absence of evidence is not the same as the evidence of absence, as stated by Daryl Bem, a psychology professor at Cornell University in an article covering this study.

Discussion Points

3. How does this evidence compare to the strength of evidence in favor of ESP?

In the described behavioral experiments, mutant males exhibited mating behaviors when in the presence of other males in a dose-dependent fashion; more mutant transporter caused more abnormal courtship. However, genderblind did not demonstrate these aberrant behaviors if sex pheromones, even male ones, were absent. In a mutant male lacking the chemical 7-tricosene, genderblind was uninterested in courtship. However, if 7-tricosene was applied to the body of the pheromone-challenged males, courtship resumed. This was especially odd since 7-tricosene was previously thought to strongly inhibit male-male courtship, an unproductive behavior for obvious Darwinian reasons. Therefore, the conclusion was that although the signal from the pheromone was detected, the processing was disrupted, causing improper circuits be activated and the wrong behavioral patterns to be initiated.

The mechanism underlying all of this altered processing falls back to the glial transporter. This protein should regulate extracellular glutamate concentration; any changes in its activity would affect synaptic transmission significantly (many studies have demonstrated the powerful role of glia in the tripartite synapse over the past few years). This assumes that the altered processing observed in genderblind is due to synaptic communication changes and not due to developmentally-induced re-wiring. To rule out the latter, the authors used genetic tricks to express genderblind in adults, after normal development of the circuits, and then induced the expression of the mutant. These flies still exhibited the same male-male courtship preferences. There are still many loose ends, not least of which is the question of which circuits actually process the pheromone signals and then translate (transmit?) the information to other (the same?) circuits governing motor programs essential for courtship behavior. Although we don’t know the answer, glia obviously play an important role in this process.

This paper, interesting from a number of angles, attempts to hype the results in a way that suggests homosexuality can be turned on or off, at least in flies, simply by using a pharmacological inhibitor of the genderblind transporter. This implies that homosexuality may be simply a result of abnormal signal processing in the appropriate circuits. This vast oversimplification is discussed in a commentary accompanying the article. Unfortunately, I’m sure that many will not read, nor even see, the thought-provoking issues raised by the commentator. This is apparent from many comments listed in two blog conversations (here and here) at the NY Times regarding these findings. This article obviously provides more than just interesting science, but also speaks to the interface between science and the public as well.

Discussion Points

4. What are the next experiments that you would do in order to advance our knowledge of pheromone processing?

Have a look and post your comments there.