ISOT 2012, dag fem

(For previous entries, see days one, two, three, and four.)


Kristin Scott:

The Scott lab focuses on taste receptors and circuits in drosophila, and today she presented two new stories.

On the receptor side, the Scott lab had identified the receptor for 3/4 of the cells in the fly taste ganglion, the SOG. Today she presented one channel for the last cell, ppk23. Another ppk, ppk25 was previously identified as a water/stretch receptor for flies. Ppk23 expression is sexually dimorphic: male flies' ppk25 cells project across the midline while females' do not; furthermore ppk23 is coexpressed with fruitless. They knocked ppk23 out, and found that male flies could no longer distinguish between males and females, and started courting male flies. Since ppk23 is in "taste" cells, they guessed it might be detecting sexually dimorphic surface hydrocarbons, and presented these hydrocarbons to the fly. Using calcium imaging they found two cells in the SOG that responded: one to male hydrocarbons, and the other to female. There are many other ppks expressed in those cells, so it's unclear whether ppk23 is the actual sensor or simply involved in signal transduction.

The second story concerned modulation of feeding behaviour. In a paper earlier this year, the found that dopamine activity  increases with hunger, and that dopamine activation can drive increased feeding behavior. Here they looked for neurons that drive satiety.  They generated GAL4 lines, and then measured how much sucrose the flies would accept. One line, 98, were "insatiable," and drank well beyond WT flies. In fact, they eagerly drank bitter fluid, or oil as well. The 98 line labelled ~30 cells in the SOG, so now they are trying to use mosaics to pin down the exact cells.

Robert Barretto:

Rob expanded on what Zuker previewed the first day: 2p endoscopy in the taste ganglion. He described seven populations: five single tastes, and two pairs, sweet/umami ans bitter/sour. Notably, umami was represented the least,  by far, with 30/1200 cells. In comparison, during the questions, he mentioned that 15-20 cells represented another pair, sour/salty (I think).
After his talk, a few people approached him with further questions. Since he did not publicly say them, I will refrain from repeating them. Suffice it to say that consideration of stimulus concentration, and crosstalk between receptors (e.g. artificial sweeteners and bitter) is essential to interpreting the results.
And with that the conference is over.

ISOT 2012, día cuatro

(For previous entries, see days one, two, and three.)

Very smelly morning, often about innate smell detection, but a significant portion of the works were already published. In the afternoon, a couple taste talks caught my eye.

Yuzo Ninomiya:

I'm a fan of Ninomiya's work, having covered his papers on leptin and cannabinoid modulation of sweet taste previously. Here, he provided an update to that work, concentrating on the relative strengths of leptin vs cannabinoids. In WT mice, cannabinoid antagonists are ineffective, while leptin antagonists are. However, in db/db mice (leptin knockouts) cannabinoid antagonists do reduce sweet responses in the CT nerve. Thus it appears that under normal conditions leptin is dominant. To verify this, they measured sweet responses in mice with varied blood leptin levels, and found that cannabinoid antagonists became more effective as leptin levels went down.

Sclafani:

Besides being sensed by the tongue, sugar is also detected by the stomach, which influences food intake over longer time scales. Sclafani's lab investigated this by directly injecting sucrose into the mouse gut (? Or IP) in T1R2 knockout mice (no canonical sweet receptor). Mice triggered the injection by licking a water spout. They employed a conditioning protocol,  where unsweetened cherry taste caused sugar water injection, while grape taste caused water injection. After conditioning, mice licked the cherry water more.

Sclafani mentioned three possible receptors: GLUT5 which can detect fructose but not galactose; and SGLT1/5 which can detect galactose but not fructose. To see which of these are involved, they switched the injection to fructose or galactose. Mice injected with fructose were not conditioned, showing GLUT5 is not responsible.  In contrast , galactose did work for conditioning.  To see whether metabolization is necessary they tried conditioning with MDG, a non-metabolizable galactose analog, and found conditioning still worked. Thus SGLT activation alone seems to be sufficient.

How could this signal downstream?  Scalfani noted that most gut hormones decrease food intake, while ghrelin, the one orexigenic hormone, is suppressed by glucose. Thus there is at the moment no clear pathway for the effect.

ISOT 2012, day 3

(For previous entries, see days one, and two.)

Lots of insect olfaction today, which is not my wheelhouse. Apparently some mosquitos are racist, and prefer to bite humans over guinea pigs, or vice versa. There were some interesting posters (e.g. ENaC knockout mice still retain some salt taste sensitivity), but it's I'm not sure how interesting brief poster summaries would be. Instead, two general points.

Zuker is well known for pushing the labelled line story from the periphery to insular cortex. In contrast every other taste researcher has found that the situation is more complicated: single cells can respond to multiple tastes on the tongue and in cortex; and some receptor knockouts can still respond to the tastes that should have been elided. So I've used this conference as an opportunity to see what other taste researchers think about Zuker's story. Their responses usually start with the phrase, "his data is beautiful but..." then give examples of results that can't fit the labeled line story. I haven't found another researcher who endorses Zuker's view, which is strange considering how high profile Zuker is. It is difficult to tell where clear-headed scientific thinking ends, and personal politics begins.

Second, while looking at posters,  I saw a number of people using channelrhodopsin by stimulating with long (>5 ms) pulses. This strikes me as incredibly imprecise, as you cannot know how the stimulated cell is firing, only that it is depolarized. Instead I'm a strong advocate of using pulsed stimulation at specific frequencies (up to 50 Hz!), so you can know exactly how the stimulated cells are firing. And to argue from authority, most high profile papers I can think of also used pulsed stimulation. People are certainly getting results with the long pulses, but I feel strange trying to interpret the results. I would be interested to know what other people who use ChR2 think.

ISOT 2012, deuxième jour

(First day's entry)


Daily blogging is rough.

This is the first medium size meeting I've been to (800 attendees, 2-3 sessions), and it is uniquely exhausting. While SFN is 40 times larger, and tiring in its sheer scope, there is something exhilarating about being surrounded by so many people, like walking through Shibuya or Times Square. If you get tired of taste or olfaction, you can take in a barrel cortex or retinal development talk. Six hours a day of chemosensory talks can be repetitive.

Since I went to so many talks today, I will briefly comment on two. The day started off with Cori Bargmann. I got in a little late, but the gist of the first part is that one of the C elegans neurons, AWC, is activated when an odor turns off (which sounds familiar to me), with a time constant of 10s of seconds. They looked at the mechanisms of this, but to be honest I got lost in the worm alphabet soup.

In the next section, they looked at behaviour. C elegans have simple motor behaviour: they can move forward, reverse, turn, or pirouette. Using a microfluidic device, they measured how the worms turned in response to odors turning on and off. They found that worms tend to move forward during odor presentation, and when the odor turned off, the worms started turning, with a time constant similar to AWC. They then tried imaging the AWC in the behaving worm, to see if AWC activity was correlated with turning or moving forward. However, that AWC was similarly active when the worm moved forward or turned, which means AWC neurons are simply sensory and not motor.

Next, she moved to the timescale of a few seconds. Worms move by wiggling back and forth in sinusoidal patterns. These sine waves in effect give the worm a metered sampling of the world, a sort of sniff. One worm behaviour that may rely on this sinusoidal sniffing is turning along odor edges; worms within an odor stripe will move parallel to the stripe, or turn inward, but not outward. Since AWC neurons fire in response to changes in odor, they tested how AWC neurons fired to stimuli at 1Hz, and found the AWC neuron could follow the stimulus. Then they performed reverse correlation on the calcium signal, using an L-N model, and found that AWC neurons could linearly follow integrate over a 1s window. (GCaMP's 0.5s non-linearity was also detected)

Jeff Isaacson:

Isaacson is looking at how experience an anesthesia can modify odor representations in the olfactory bulb. They now have 2p awake imaging working in the bulb, by injecting AAV-flex-GCaMP in Pcdh21-cre mice. In slices, they validated that GCaMP could encode APs linearly over 0-40Hz.

Using this, they imaged responses in the bulb. They found that in awake mice, individual cells responded to a relatively narrow range of odors (imaging over 4s, ignoring sniff phase). When they recorded in anesthetized mice, the responses increase in both magnitude, and broadness. They also lost inhibitory odor responses. They hypothesized that anesthesia might be selectively effecting GABAergic granule cells, and so applied gabazine in awake mice, and saw a similar effect to anesthesia. To look at this another way, they then imaged activity in the granule cells using GAD-cre mice with AAV-flex-GCaMP. The granule cells had sparse tuning. However, when they anesthetized these mice, the found the granule cells simply stopped responding to odors, which would explain the loss of inhibition.

In the 2nd half of the talk, he asked the question of how stable odor representations are. They imaged mice daily over 7 days, and found the response magnitudes decreased each day. Then they split their odor space in two; for group A, they imaged those odors every day; for group B, they imaged only on days 1 and 7. The group A responses all decreased, while the group B responses maintained their strength. Thus repeatedly presenting an odor selectively attenuates its response. This also held for single cell analyses. To look at glomerular inputs, they imaged OMP-SpH mice, and found that these responses were maintained over 7 days. They looked at how long it took for these responses to recover, and found that they began to recover after one week, but took a full 2 months to completely recover. Finally, they looked at how awareness effected this by imaging mice in both awake and anesthetized states. While the awake responses decreased, in the same mice, the anesthetized responses remained stable. Thus, the adaptation they observe in awake mice requires some sort of awareness.

ISOT, 1日

Perk of attending chemosensory conferences: the candy.

The conference kicked off with a keynote speech by Charles Zuker. I've written about Zuker's work multiple times before (lack of links due to phone posting). His talk roughly split into five sections, spanning from taste receptors to the amygdala. In the first and third sections he briefly reviewed work on taste receptors and cells, and his lab's recent paper on taste "hotspots" in cortex.  As these have been published,  I will focus on the three sections where he presented unpublished data.

In the second section, he presented recordings from the taste ganglia. They stuck a 2p-endoscope into the taste ganglia to see what combination of tastes the neurons responded to. Assuming only five taste modalities (sweet, sour, salty, bitter, and umami, and ignoring carbonation or fat), there are 31 possible combinations of taste receptive fields, including pairs, triplets, quadruplets, and the full taste house. Using a "magic" dye, they found only seven of these combinations, the five basic tastes, and two pairs (the slide went by before I could note both). Zuke breezed through this quickly,  as the researcher will present later in the conference.

Zuke seems quite enthusiastic to pursue taste into "higher" brain regions, which he showed in the last two sections. Having described taste hotspots in gustatory cortex, he wanted to show they were necessary and sufficient. To show they are necessary, the lab trained mice in a go/no-go task where the go cue was a sweet taste, and the no-go was a bitter taste, and the task was to lick (or not) following the cue . Thirsty mice were rewarded with water. Then the lab bilaterally cannulated the mice in either the sweet or bitter hotspot, through which  they injected a "neuronal silencer." When they injected in the sweet area, the mice failed to stop licking  during the no-go trials, showing they may not have sensed sweet. Conversely, when they silenced the bitter area, the mice did not lick for the go cue. Thus they showed that these areas are required for sensing their respective modalities.


To show the areas are sufficient for tasting these modalities, they employed Channelrhodopsin. First, the bitter area. They infected the bitter hotspot with AAV-ChR2-YFP. They then water-deprived mice, and trained them to lick a water "sipper." The mice would lick freely, until they turned on the light in the bitter hotspot, which caused the mouse to wince and stop licking. This could be turned on and off for a few seconds at a time, showing the effect was nearly instantaneous. And if they turned the "power" of the light up (unclear whether intensity or frequency), the mice would wince and gape from the awful sensation.

For the sweet hotpost, they trained mice to occasionally lick a water spout. Since they didn't want the mice licking for liquid reward, they used slaked mice. For some of the licks, the licks would trigger the light in the sweet hotspot; for other licks, it would trigger nothing. They then measured the number of licks when the light was functional versus not, and found the mice licked more when the licking triggered the light.

In the last section of the talk, Zuker focused on the downstream areas from gustatory cortex. They have injected AAV-GFP and -tdTomato in both the sweet and bitter hotspot, and identified four downstream areas: amygdala, hypothalamus, entorhinal cortex, and the nucleus accumbens. The labeling in the amygdala did not label the entire amygdala, but one sub-field.

I'm not an expert in amygdala function, but it is involved in valence. For unclear motivation, they infected the sweet hotspot with AAV-ChR2, and stuck a light fibre in the amygdala. They then stimulated the sweet axon terminals in the amygdala while the mice were tasting a bitter compound. When the light was on, the mice licked frequently; however, with the light off, the mice did not lick. The effect of light could also be blocked by NBQX.

For future directions, Zuker basically highlighted most interesting questions in central taste. How are mixtures encoded? How does internal state (i.e. satiety) alter circuits? How are olfaction and taste integrated as flavor? And they now have chronic 2p endoscopes working in behaving mice.

All in all, I nice overview of many exciting experiments. One large outstanding question remains from before: given that all e-phys recordings have shown that neurons are broadly tuned, why hasn't Zuker's lab been able to replicate that (Zuker notably presented almost all imaging, and one e-phys slide regarding a ChR2 control). More generally, it will be fun to look at these results in a complete paper. Right now it's not clear to me that the cannula and light fibre experiments are specific enough to make conclusions about bitter versus sweet taste modalities, or whether it is simply aversive versus pleasant. And the amygdala experiment seems more like a fun pilot experiment rather than a complete story. In any case, it's nice to see a big lab presenting unpublished data to a wide audience.

Taste coding in mammals

Aside from the main talks, ISOT runs three parallel sessions at a time. Since I want to work in taste, I will focus on the taste sessions, including this afternoon's session on taste coding from the periphery to central areas.

First up was G. Hellekant, who presented some simple experiments recording from taste nerve fibres. He started by showing that mammalian taste receptors can vary 50-70% in homology between species. Specifically, ruminant mammals are quite different from primates. Then he showed taste nerve recording from chimpanzees and marmosets, that showed the responded to a wide variety of taste modalities, from sweet to bitter. In contrast, cows and pigs responded to a smaller subset, notably lacking many sweet responses.

In the second half, he mentioned two small facts. First, there is a chemical, gymnemic acid, which blocks sweet receptors in humans (lactisole also does the same). Second, he showed that a calcium channel, Calhm, is expressed in T1R2 cells, and that Calhm KO mice do not have a sweet response.

The second speaker was Susan Travers, who focused on taste responses in the NST and PBN of anesthetized rats. Previously, she had reported that there are four clusters of taste responses - Sweet, NaCl, Acid, and Bitter - based on their average firing rate during 20s of taste presentation. However, recognizing that taste responses are dynamic, she focused on the temporal aspect of the responses. She showed that during the first second of the response, many cells were broadly tuned, responding to multiple tastes. Notably, sweet cells responded quickly, while bitter cells took some time to ramp their response.

To look more closely at the temporal information, she performed a metric space analysis, which I did not quite follow. From this, she concluded that including temporal information increases total information, which seemed obvious from simply looking at how dynamic the responses were. Also of note is how slow these responses are developing over seconds, when a lick is 150ms. In any case, it appeared that a subset of PBN neurons were still broadly tuned.

Third up was Sid Simon, whose main point was that taste neurons can have highly diverse responses, both narrowly and broadly tuned. He showed recordings from rats with electrodes implanted four places: OFC, insular cortex (IC), nucleus accumbens (Nacc), and amygdala. These rats were then trained in a go/no-go task. Each of the recorded places had special kinetics. IC neurons responded to licking; OFC neurons anticipated the cue; amygdala neurons responded to the reward. For IC neurons, some were inhibited by feeding, while other were excited or unchanged; and these same neurons would become inhibited, excited or unchanged during sleep.

In the last section, he showed data from a rat trained to discriminate between different concentrations of NaCl. 60% of IC cells didn't respond to the taste of NaCl. Of those that did respond, the responses were dynamic. Some responded during the first lick before fading, while others increased their response over time. Some just responded to water, but not NaCl; and of those, some could differentiate between reward water, and cue water. All in all, if you can imagine a taste receptive field, some neuron probably has it.

The fourth speaker, Claire Murphy, presented fMRI data on satiety. I'm not a big fan of fMRI, but her main point was that hunger increases the magnitude of taste responses in IC, OFC, amygdala, caudate, etc.; and that some areas do not respond in sated humans, but only when they're hungry.

That's it for day one.

Live, from Stockholm, it's ISOT XVI!

I will be presenting a poster on Sunday, June 24th at ISOT (#167), which I have uploaded the poster to figshare. The first half of the poster should be old hat for avid blog followers, but there's super-sekrit, new data in the last column. If you have any comments about the poster, my research, or the blog, please stop by to chat.

Abstract for the poster:

Mitral cells in the olfactory bulb of mammals encode odors dynamically via short, phasic responses throughout the breathing cycle. Less attention has been paid to how dynamic these responses are between breaths.

To look at the inter-breath dynamics of odor coding, we performed multielectrode recording from the olfactory bulb of awake (and anesthetized) head-restrained mice. We found that the odor code is not stationary, and that individual cells change their response between breaths. On the population level, we show that the first breath is different from each subsequent breath.

We also looked at how odors are coded after the end of odor presentation. We found that individual cells have post-odor responses that can last for seconds. On the population level, this information contains odor- and concentration-specific information. Increasing the length of odor presentation caused longer post-odor responses. To investigate the possibility of network reverberations, we artificially stimulated the olfactory bulb of Thy1-ChR2 mice, and found that stimulation could cause persistent activity for seconds following stimulation. Finally, to show that feedback can come from higher areas, we measured the LFP in the OB of mice with the ipsilateral OB blocked, and found there was still activity.

Trail Tales: Jose Esteban, and PI3K signaling in synaptic plasticity

Jose Esteban (formerly of Michigan, now at Madrid) gave a talk today about intracellular trafficking, PI3K signaling, and Alzheimer's disease. I'm going to a few conferences this summer, so I'm going to use this as test-run for limning talks. Two-thirds of the talk was unpublished data, so I will try to paraphrase the results in figure form (parafigure?).

Most of the seminar was about AMPA receptor (AMPAR) trafficking, so a quick refresher. AMPAR are the most common ionotropic, excitatory glutamate receptor in the brain, and it is thought that learning and memory comes from changing the number of AMPAR in synapses, viz. synaptic plasticity. Some stimulation protocols can lead to an increase in AMPARs at the synapse, called long-term potentiation (LTP), while other stimulation protocols remove AMPAR from synapses (long-term depression, LTD). Whence and whither do the AMPAR go from the synapse? From early endosomes and to recycling endosomes. All of this transport is controlled by a large web of signaling proteins, including, for today, PI(3,4,5)P₃-kinase (PI3K). PI3K activity has been shown to be involved in LTP, while PIP₃ phosphatases are involved in LTD.

Rab trafficking


As mentioned above, AMPAR are trafficked through various endosomes. The endosomal targeting is controlled by, in part, Rab-family GTPases. Each Rab GTPase can target a given piece of membrane towards a specific endosome. The important Rab family members today are Rabs 4, 7, and 11, outlined below. (All figures were made by me, and represent my impressions from the talk. If I got something wrong, holla at me.)

AMPAR trafficking in endosomes is regulated by the Rab GTPas family. AMPAR at the synapse (top) are internalized via Rab5, then transferred to another endosome (early?) by Rab4. From there they are sorted to the lysosome for degradation by Rab7 (grey oval), or to back towards the synapse by Rab11.

To look at AMPAR trafficking, the Esteban lab performed chemical LTD (cLTD), and measured two parameters. First, they simply measured the synaptic AMPAR current. Second, they looked at AMPARs directly by transfecting organotypic hippocampal slices with GFP-GluR2. Then to look at whether GluR2 was expressed at the plasma membrane, or in endosomes, they stained for surface GluR2. (I'm disappointed they didn't use SEP-GluR2) Under control conditions, following cLTD, they saw a decrease in synaptic current, and a decrease in both surface and total AMPAR in spines.

LTD (and thus AMPAR trafficking) is regulated by various Rab GTPases. During normal LTD, you get a decrease in synaptic current of ~50% (not to scale). With either a Rab4 or Rab11 DN, LTD is increased. With a Rab7-DN, LTD is decreased.

They then proceeded to disable individual Rab proteins by transfecting slices with dominant negative (DN) Rabs. When the transfected Rab4-DN, they found that the synaptic currents were decreased even more than control. By measuring AMPAR localization, however, they found that while the surface AMPAR level was decreased like control, the total AMPAR level in the spine was the same. This means that some AMPAR were internalized, but not properly distributed.

To look more directly at lysosomal degradation, they performed cLTD in the presence of a protease inhibitor, leupeptin, which should block lysosomal degradation (Esteban said this was the first time this simple experiment was done.). However, leupeptin had no effect on the synaptic currents measured.

To look at the role of the lysosome another way, they transfected slices with Rab7-DN, which would prevent AMPAR from being transported to lysosomes. In Rab7-DN cells, cLTD was in fact decreased, in contrast to the lack of effect of leupeptin. From this they hypothesized that once internalized, AMPAR can go two ways: to the lysosome, or back to the plasma membrane. If you block the lysosomal pathway, they then must go back to the PM. However, this means that the actual degradation itself is inessential for LTD. As a final experiment, they transfected cells with Rab11-DN, and found that this increased LTD.

PTEN and Alzheimer's

In the middle section of the talk, Esteban described two sets of experiments he published previously. Arendt et al, 2010 covered PI3K's role in AMPAR trafficking during LTP, which I covered in the very first post on this blog o' science. Jurado, et. al., 2010 showed that the PIP₃ phosphatase, PTEN, is essential to LTD.

In the last third of the talk, Esteban presented new work on Alzheimer's. One facet of Alzheimer's disease is that synaptic plasticity is altered, and in simplistic terms, may favor LTD over LTP, leading to a loss of spines. Since Esteban has been investigating PI3K and PTEN, he asked whether altering PTEN function could alleviate some of the symptoms of LTD.

Mice don't normally get Alzheimer's, so to generously give it to them, Esteban's lab transfected slices with amyloid precursor protein (APP). They then performed LTP on these slices, and found that APP almost completely abolished LTP. Then to see if PTEN could effect this, they bath applied a PTEN inhibitor, and found that it could partially rescue LTP.

PTEN inhibition rescues LTP and memory. A. Vehicle, or PTEN (i.e. PTEN inhibitor, sorry for the label), allows normal LTP. APP almost completely blocks LTP. PTEN inhibitor is able to rescue LTD. B. In either an object movement or contextual fear conditioning test, PTEN inhibitors are able to rescue the APP phenotype.

Having seen the effect of PTEN inhibitors on LTP, they next asked if it could effect behaviour. They implanted rats with a minipump full of the PTEN inhibitor, and performed two memory tasks. First, they employed an object movement task, where mice were familiarized with objects in a cage. One of the objects was moved, and the mice were tested to see if they noticed. Second, they used good old contextual fear conditioning. In both of these tasks, memory was impaired by APP (or perhaps a different Alzheimer's model, my notes are unclear), but rescued by PTEN inhibitor.

Question time


I slacked in note-taking during the discussion, which is probably one of the more interesting parts of a talk for strangers. Here are two points I salvaged.

Esteban mentioned that PTEN, given its influence on PIP₃, can effect a wide variety of systems besides simply effecting synaptic plasticity, so more work would need to be done to understand how this works. (Indeed, given PI3K is important for LTP, it is unclear to me whether this is a downregulation of LTD, or an indirect upregulation of LTP. In either case, the balance appears to be changed.)

Second, it is not clear why PTEN had no effect when applied alone. One might expect PIP₃ to accumulate, which should effect something. Esteban noted that there are ubiquitous feedback systems, which could perhaps compensate for a loss of PTEN function.