Oh, to regenerate axons! Hypoxia inducible factor’s role in axonal regeneration

How does the nervous system respond to axonal injury? The answer is: it depends. In the environment of the central nervous system (CNS), neurons possess extremely low regenerative capacity. In contrast, the peripheral nervous system’s (PNS) neurons exhibit the ability to regrow following injury. While the mechanisms underlying this regrowth is not completely understood, there is significant motivation to elucidate this field for the purpose of developing therapies to enhance axonal regeneration even in the inhibitory environment of the CNS.

Dr. Valeria Cavalli’s group at Washington University decided to study axonal regeneration in the context of the dorsal root ganglion (DRG). What makes the DRG neuron an interesting model system is its structure as a pseudounipolar neuron: from the cell bodies located in the dorsal root ganglion located adjacent to the spinal cord arises a single extension which splits into two branches: a central branch that navigates back to the spinal cord, and a peripheral branch, which as the name suggests projects peripherally. Injury to this peripheral branch of the DRG results in activation of a pro-regenerative program – that is, a series of molecular events that promote the regrowth of the damaged axon. The central branch, keeping in theme with the CNS, does not exhibit such regenerative capacities. What, then, allows a single DRG neuron to exhibit repair on one branch but not the other?

Transcriptional profiling studies in DRG neurons identified a handful of interesting genes upregulated following injury; among these is the gene known as Hypoxia Inducible Factor (HIF), a heterodimeric protein made of HIF-α and HIF-β subunits. HIF-α has a particularly interesting expression profile: at physiologic O2 concentrations, HIF-α is targeted for ubiquitination/degradation pathways. As O2 concentrations drop, this suppression of HIF-α expression is released.

Cho et al., 2015, showed that following peripheral DRG axotomy, a large percentage of HIF-α targeted genes are upregulated over a 1.2 fold threshold; furthermore, this upregulation of gene expression is lost in HIF-α knockdown DRGs. In uninjured DRG neurons, constitutive overexpression of HIF-α, but not knockdown of HIF-α, lead to significant changes in gene expression compared to wildtype uninjured DRG neurons.

To study the regenerative capacity of DRG neurons, Cho et al. infected DRG neurons either with control shRNA, two HIF-α targeting shRNAs, or a lentivirus virally overexpresses HIF-α. These neurons were axotomized and immunostained for SCG10, a marker of axon regeneration. Interestingly, both shRNA-mediated knockdown and lentiviral overexpression of constitutively active HIF-α resulted in reduced SCG10 fluorescence, suggesting that regulated expression of HIF-α (as opposed to constitutive overexpression) is necessary for axonal regrowth. Further downstream, Cho et al. conducted knockdowns of genes targeted by HIF-α, and showed that while knockdowns contributed to reduced axon regeneration for some genes, there were still others genes that, when knocked down, instead exhibited slight increases in axon regeneration. This would suggest that HIF-α targeted genes could either promote or inhibit axon repair.

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In order to show how HIF-α regulates axon regrowth in vivo, Cho et al. generated mice lacking HIF-α in sensory neurons by crossing HIFAflox/flox with mice expressing Cre under the Advillin promoter (HIF1AcKO), which is specific to the peripheral sensory nervous system. After confirming HIF-α knockout, the experimenters crushed the sciatic nerve and quantified regeneration of the nerve axons 3 days later by SCG10 staining. Using SCG10 intensity and distance from crush site, the researchers calculated a regeneration index; in the HIF1AcKO, regeneration was limited past the crush site (Fig 4A-C). Injury to the sciatic nerve, but not the dorsal root itself, results in elevated HIF-α expression (Fig 4D,E).

While hypoxia does increase HIF-α levels in vitro, Cho et. al specifically showed that acute intermittent hypoxia (AIH) enhances axon regeneration in vivo via elevating HIF-α and subsequent downstream target genes such as vascular endothelial growth factor (VEGFA). They demonstrated axon regeneration both in the sensory sciatic nerve fibers as well as in sciatic motor neuron: sciatic injury in YFP-16 mice, which express yellow fluorescent protein (YFP) in motor neurons, showed increased colocalization of YFP and neuromuscular junction boutons following AIH treatment for sciatic nerve injury. Taken together, these results suggests that AIH may drive axon regeneration in the periphery via HIF-α-dependent mechanisms.

To learn more about the work conducted in Dr. Cavalli’s lab, attend her talk this Tuesday, March 21th, at 4:00pm in the Center for Neural Circuits and Behavior.

Junmi Saikia is a third year MD/PhD student in the Malinow Lab at UCSD. Her research interests revolve around neurodegenerative diseases.  She apologizes in advance  for the punishably punfunny title.

Ripples in time: How the hippocampus helps us learn, not just during sleep

We’ve all at some point been told that sleep is essential for our memories to be consolidated: when we sleep, our hippocampus takes all of the things we’ve learned during the day and files them away for easy access when we’ll need them next. Known contributors to this phenomenon are Sharp-wave-ripple events, or SWRs, are a brief (lasting only 100-200ms) oscillation of hippocampal neural activity occurring at approximately 200Hz. Interestingly, they occur not only during slow-wave sleep, but also during awake stillness—and although their presence during sleep has frequently been attributed to the memory consolidation process, their function during awake states is unclear. What role might SWRs play while we’re awake, and how does their occurrence affect our behavior?

Dr. A. David Redish at the University of Minnesota is interested in precisely these questions. His lab combines the use of multi-electrode recordings with computational analysis that allow for the study of neural ensemble activity at high temporal resolution. A recent (2016) paper from Dr. Redish’s lab, titled “Interplay between Hippocampal Sharp-Wave-Ripple Events and Vicarious Trial and Error Behaviors in Decision Making”, elucidates a possible role for sharp-wave-ripple events in the context of decision-making.

Redish and his group quantified the presence of Vicarious Trial and Error (VTE) behaviors—a measure of uncertainty and deliberation—in relation to the presence of SWEs captured through neural recordings as rats completed several complex navigation tasks to obtain rewards. These behavioral tasks enabled the experimenters to measure the rats’ learning, deliberating, and decision-making in the context of SWE occurrences. The experimenters were ultimately able to observe an association between SWEs in awake states and VTE behaviors as rats decided which path to take in the tasks.

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Part A in the figure above demonstrates the nature of the spatial alternation task. A1 and A2 comprise the first trial, in which the rat had to exit the center arm and move to a side arm to obtain a reward, then return to its starting position in the center arm to obtain a second reward. The second trial, in A3 and A4, shows that the rat needed to travel to the opposite side arm from the first trial (and back to center) in order to obtain the rewards. Part B illustrates a key finding of Dr. Redish’s paper: that the disruption of hippocampal sharp-wave-ripple events (SWR) increased the proportion of VTE behaviors.

The results of Dr. Redish’s study suggest that SWRs and VTE behaviors are inversely correlated. Moreover, because VTE behaviors are associated with increased behavioral uncertainty and variability, they imply that SWRs and associated mechanisms engaged in learning and memory might play a specific role in decision-making once a particular behavior or reward has been learned. Thus, there is clearly a complex interplay of these two processes occurring during awake learning and navigation that demands exploration in greater detail.

To learn more about the work conducted in Dr. Redish’s lab, attend his talk this Tuesday, March 7th, at 4:00pm in the Center for Neural Circuits and Behavior.

Marley Rossa is a first-year Ph.D. student whose research interests encompass the use of computational techniques to analyze neural data and thereby understand the nature of information communicated between neurons and neural ensembles.

 

 

Divide and Conquer! How the Brain Avoids Sensory Overload

They say that he human brain is the most complex machine in the universe, and it’s for good reason. Brains are comprised of about a hundred billion neurons that typically form a thousand unique connections each. As a result scientists estimate that the brain can store ten to a thousand times more information than modern day laptops.

computer-brain-interface-darpa-sHowever, what is even more impressive than the storage capacity of the brain is its ability to dynamically read and process massive amounts of information. Every second that you are awake, your brain is efficiently parsing through constantly flowing streams of visual, auditory, gustatory, and olfactory data. How does the brain sort through all this rich information? What mechanisms underlie our ability to prioritize important sensory information when presented with competing or conflicting cues?

These open questions in neuroscience have long puzzled scientists. In this post, we will take a look at the paper from Michael Halassa’s group, titled “Thalamic control of sensory selection in divided attention”,  that sheds some light on these questions. The central goal of the article is to provide some proof for the hypothesis that the frontal cortex, the home of executive function, regulates the selection of sensory information in the thalamus, the hub of sensory inputs into the brain.

To accomplish this, Halassa first creates a rodent psychometric test that basically tests the ability of a mouse to secure a food reward when presented with either congruent or conflicting visual and auditory stimuli.  As expected, mice were forced to divide their attention when presented with both visual and auditory information. When presented with conflicting auditory tones, mice had a higher visual detection threshold, which persisted even as the tones were switched back to being congruent. This provided evidence that top-down processing, likely from the frontal cortex was biasing the visual stream! Next the authors sought to pin down the circuit mechanisms of this finding.

Next using optogenetics, a technique to make neurons respond to light, Halassa’s group linked channelrhodopsin to GABAergic inhibitory cells in the frontal cortex. This allowed them to effectively silence activity between a region of the frontal cortex that sends projections to the thalamus, the sensory hub. In the same behavioral tests, the mice perform much worse when the link between the frontal cortex and the thalamus is inhibited. Without this connection, mice are suddenly unable to sort out conflicting sensory cues in order to complete the task!screen-shot-2017-03-06-at-1-30-21-am

Importantly, the paper also shows that a similar sort of disruption scheme in the visual cortex fails to disrupt task performance in a similar manner. This means that the cross-cortical interactions are less important than corticothalamic connections, which was previously not known.

In order to fully characterize the circuit, optogenetic methods were combined with viral tracing to follow the projections from the frontal cortex to a particular region in the thalamus, called the thalamic reticular nucleus (RTN). Using electrophysiology and optogenetics, it is shown the neurons in this region fire specifically when conflicting auditory tones are played, and that silencing this region results in the a similar hit in task performance, as above. Quite convincingly, it seems the the thalamic target of the frontal cortex for sensory selection is the RTN.

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To wrap this study up, the authors investigated the interaction between the RTN and adjacent lateral geniculate nucleus (LGN), a thalamic structure that is crucial for visual processing. Using a novel assay for in vivo inhibitory activity (FRET photometry), they demonstrate that when mice are attending to conflicting auditory cues, the RTN uses direct feed-forward inhibition, as measured by intracellular chloride ion shifts, on the LGN to alter visual perception.

In summary, this article elegantly describes the link between the frontal cortex, the thalamus, and the visual system using a plethora of high-powered and novel experimental techniques. The authors provide convincing evidence for a mechanism in which the more “intelligent” regions of the brain regulate the flow of sensory information such that cues relevant to the task at hand are amplified and useless cues are discarded.

To learn more about the work conducted in Dr. Halassa’s lab, please attend his talk this Tuesday, March 7th, at 4:00pm in the Center for Neural Circuits and Behavior.

Debha Amatya is a third year MD/PhD student in the Laboratory of Genetics at the Salk Institute. He’s interested in psychiatric genomics and weightlifting. 

Optogenetic regulation of dopamine neurons in the midbrain regulates depression-related behaviors in mice

In 2015, 4% of U.S. adults aged 18 or older experienced a serious mental illness that interfered with daily functioning, and this percentage increases to almost 18% when adults with any mental illness are considered. Major depression in particular is one of the most common psychiatric disorders in the U.S., and has the highest disability burden among all mental disorders. Despite the social and economic costs of depression, relatively little is known about its neurobiological basis.

Anhedonia, or the inability to experience pleasure from activities usually found to be enjoyable, is a key feature of depression, and suggests that there may be a deficit in the reward circuitry of the brain in this disorder. Dopaminergic neurons in the ventral tegmental area (VTA) of the midbrain that project to the nucleus accumbens (NAc) have been implicated in reward recognition and initiation of reward utilization. Alterations in the function of this pathway could potentially underlie some of the behavioral deficits seen in people with depression.

Two in vivo firing patterns have been identified in dopaminergic VTA neurons: 1) low frequency tonic firing, and 2) high frequency phasic firing. Previously, phasic firing was shown to be upregulated by repeated social-defeat stress (a mouse model of depression) in susceptible mice, whereas mice resilient to the effects of repeated social-defeat showed no firing rate change.

Dr. Eric Nestler’s lab at Icahn School of Medicine at Mount Sinai in New York used optogenetic techniques to drive different firing patterns in dopaminergic VTA neurons in order to assess whether phasic firing patterns could induce depression-like behaviors in mice. They injected double floxed Cre-dependent AAV virus expressing channelrhodopsin-2 fused with enhanced yellow fluorescent protein (ChR2-eYFP) into the VTA of tyrosine hydroxylase (TH)-Cre mice to target expression of ChR2-eYFP to dopaminergic VTA neurons. VTA neurons were driven via activation of ChR2 at either a low frequency tonic pattern or a high frequency phasic pattern during a subthreshold social-defeat stress paradigm. Mice that received phasic stimulation showed a significant increase in depression-like behavior compared to mice receiving tonic stimulation. The depression-like phenotype was characterized by decreased social interaction with another mouse and decreased sucrose preference.

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Chaudhury et al. (2013) then assessed whether phasic firing in VTA neurons could confer susceptibility in mice resilient to the social-defeat paradigm. A standard 10-day social-defeat paradigm was performed in wild-type mice, and a social-interaction test was then performed to identify resilient mice. HSV-ChR2-eYFP virus was injected into the VTA of resilient mice, and a second social-interaction test was then performed while undergoing optogenetic phasic stimulation of VTA neurons. The behavioral phenotype of these mice switched from resilient to susceptible, as shown by a decrease in social interaction and a decrease in sucrose preference 12 hours after optogenetic activation of VTA neurons. VTA neurons exhibited sustained alterations in excitability 12 hours after optogenetic activation, evidenced by increased spontaneous and evoked activity. This increased excitability may explain the decreased sucrose preference seen 12 hours after VTA neuronal stimulation in the social-interaction test.

Given that VTA dopamine neurons project to numerous brain areas, they wanted to test the role of the VTA-NAc pathway specifically in mediating the depression-related phenotype. Replication-defective pseudorabies virus expressing Cre (PRV-Cre) was injected into the NAc, and the Cre-dependent AAV ChR2-eYFP virus was injected into the VTA. This combination caused expression of ChR2 in only NAc-projecting VTA neurons. Mice were subjected to the subthreshold social-defeat paradigm, and then 24 hours later optogenetic stimulation of VTA neurons was performed during the social-interaction test. This manipulation reliably induced the depression-related phenotype of increased social avoidance and decreased sucrose preference. Similar methodology was employed when testing the effects of inhibiting this pathway, but an AAV halorhodopsin-eYFP virus was injected into the VTA instead. Inhibition of VTA neurons during the social-interaction test induced resilience to the depression-related phenotype, with increased social interaction and increased sucrose preference.

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Manipulation of firing rates in VTA neurons projecting to the medial prefrontal cortex (mPFC) were also performed in this study using ChR2 and halorhodopsin, and the same methodologies that were used to study the VTA-NAc pathways were employed. Optogenetically induced phasic firing of VTA-mPFC neurons had no effect on social interaction and sucrose preference after subthreshold social defeat. Inhibition of VTA-mPFC neuronal activity via halorhodopsin activation during the social-interaction test induced a susceptible phenotype (decreased social interaction) after subthreshold social defeat, but no difference in sucrose preference was observed.

The results of these studies delineate neural circuitry involved in the induction of depression-like behavior after social-defeat stress, specifically the VTA-NAc pathway mediating anhedonia-like behavior. As we begin to unravel the neurobiological complexity of depression and other psychiatric illnesses, new therapeutic strategies can be created to more selectively target pathways responsible for the behavioral abnormalities seen in these disorders.

To learn more about the work conducted in Dr. Nestler’s lab, please attend his talk this Tuesday, February 28, at 4:00pm in the Center for Neural Circuits and Behavior.

Molly Kwiatkowski is a third year MD/PhD student in the Young lab located in the Consortium for Translational Research in Neuropsychopharmacology. Her work focuses on elucidating neural mechanisms underlying psychiatric disorders such as bipolar disorder.

 

Analyzing Associations: Differential Amygdalar Activity to Conditioned Stimuli

Associative learning, the process by which an organism forms a link between a cue or behavior and an intrinsically valenced stimulus, is a critical feature of adaptive behavior and survival. Learning to run upon hearing a roar associated with a predator allows one to avoid getting maimed or eaten; learning to rush to the kitchen when you smell freshly baked cookies enables you to snag a warm, tasty treat. As demonstrated by these two examples, associations can occur with both negatively valenced and positively valenced stimuli, with the former often invoking withdrawal and avoidance behaviors and the latter enforcing active approaching behaviors. Because associative memory acts as such a cornerstone in shaping flexible and appropriate behaviors, when this process goes awry, various negative consequences can ensue: depression, substance abuse, and anxiety disorder are only a few of such maladies tied to disturbances in circuitry underlying associative and motivational functionality. Consequently, studying this subject is of paramount therapeutic, as well as intellectual, interest.

Dr. Kay Tye’s Lab at the Massachusetts Institute of Technology aims to understand these very processes, employing a variety of methods from optogenetics and electrophysiological recordings to pharmacological and imaging techniques. A recent paper published by Dr. Tye and colleagues provides significant insight into the routing of positive and negative information from the amygdala during memory retrieval, suggesting that specific cell populations in the basolateral amygdala (BLA) encode different types of emotional valence depending upon the region to which they project. While the BLA as a whole is crucial in forming both positively and negatively valenced associations, subpopulations within the BLA demonstrate distinct activity patterns to rewarding versus aversive conditioned stimuli.

The main targets of the BLA investigated in Beyeler et al.’s (2016) study included the nucleus accumbens (NAc), central amygdala (CeA), and ventral hippocampus (vHPC). While the NAc is generally involved in positive reinforcement, the CeA is linked to learning that mediates aversive behaviors, and the vHPC presumably promotes anxiogenic actions. To examine the neural codes of BLA neurons targeting the NAc, CeA, and vHPC, Beyeler et al. (2016) utilized a dual-virus approach entailing optogenetic-mediated phototagging and in vivo electrophysiological recordings in mice trained to associate a particular tone with sucrose (a rewarding outcome) delivery and another tone with quinine delivery (an aversive outcome). After the mice learned the associations between each stimulus and its respective outcome—assessed as showing anticipatory licking selectively after hearing the sucrose-predictive tone for 70% of the trials—Beyeler et al. (2016) performed acute recordings in the BLA.

Half of the neurons recorded in the BLA were found to respond to sucrose and/or quinine, with 28% of neurons demonstrating selective responses to the tone associated with sucrose and a mere 9% altering firing selectively to the tone associated with quinine. In addition, 13% of BLA neurons responded to both tones similarly, and less than 1% responded to both tones in qualitatively different manners.

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Nevertheless, the most notable finding relates to the analysis of the subpopulations in the BLA that responded to both or to either of these auditory cues. Specifically, the subpopulation of BLA neurons that projected to the NAc exhibited a starkly different response profile than that of the BLA-CeA neurons. Zero BLA-NAc neurons were found to be excited by the tone associated with quinine, but nearly all (77%) were excited by the tone tied to sucrose; Any BLA-NAc neurons that reacted to the quinine-associated tone demonstrated inhibition by the conditioned stimulus. In contrast, all BLA-CeA projectors recorded were excited by the tone paired with quinine.

Classifying BLA projector populations using “valence bias”—considering excitation and inhibition as qualitatively different—reinforced the differential activity of the three subpopulations examined. Approximately half of the BLA population encoding valence showed a positive bias—increasing firing for the sucrose-associated tone and/or decreasing activity to the quinine-associated tone—while the other half appeared to encode negative valence—increasing firing to the quinine cue and decreasing firing to the sucrose cue. Within the BLA, a greater percentage of BLA-NAc projectors were observed to be encode positive valence (80%, compared to 31% in the BLA-CeA); whereas more BLA-CeA cells encoded negative valence (69%). Interestingly, the BLA-vHPC subpopulation responded evenly to both tones, with 43% encoding positive valence.

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These findings clearly suggest that BLA-NAc neurons preferentially encode cues associated with rewarding outcomes, consistent with the known role of the NAc in positive reinforcement; and that BLA-CeA neurons preferentially encode cues linked to aversive outcomes, also consistent with its acknowledged role in assigning negative valence to stimuli. The observation of BLA-vHPC responses evenly distributed between positive and negative valence is somewhat surprising due to its supposed role in anxiogenic behavior and requires further investigation.

If you find this experiment intriguing and would like to learn more about the nuances of and mechanisms underlying neural systems supporting associative learning, motivation,  and emotional processing, attend Dr. Kay Tye’s talk this Tuesday, February 21, at 4:00 pm at the Center for Neural Circuits and Behavior.

 

Gina D’Andrea-Penna is a first-year neurosciences Ph.D. student whose interests largely reside in the domain of cognitive neuroscience, particularly in investigating perceptual awareness, attention, and working memory.

 

Using Optogenetics to Shed Light on Deep Brain Stimulation

Medical interventions often come about by the application of current theory surrounding a disease, but ultimately whether one is approved and used in patients depends not on whether it fits with current dogma, but simply on whether it works. Consequently, as the theories surrounding how a disease develops change with new research, studying why certain treatments are effective can provide further information about the disease’s pathophysiology. In addition, research into how a treatment exerts its effect can reveal avenues to refine and improve the intervention, as well as generate new and better strategies to tease apart and understand complex biological systems.

Dr. Viviana Gradinaru’s lab at Caltech uses new techniques in neuroscience such as optogenetics, CLARITY, and 2-photon imaging to study deep brain stimulation (DBS), which is an effective treatment for various neurological disorders. As a postdoctoral researcher working with Dr. Karl Deisseroth at Stanford, Dr. Gradinaru used the then-developing technique of optogenetics to study how DBS of the subthalamic nucleus (STN) improves the symptoms of Parkinson’s disease. Optogenetics is a technique where light-activated channels are expressed in neurons and used to influence the behavior of those neurons. These channels can also be targeted to specific populations of neurons using those neurons’ genetic markers, making this technique especially powerful.

The researchers used optogenetics to attempt to identify which populations of cells contribute to the efficacy of DBS in the STN. First, they created mice that model Parkinsonian symptoms by using a chemical called 6-OHDA to kill most of the dopamine neurons in the substantia nigra pars compacta (neurons which are lost in Parkinson’s disease). Unlike in Parkinson’s disease, however, the neurons were killed only on one side of the brain, causing deficits only on the opposite side of the body. The severity of the mice’s parkinsonism could then be assessed by measuring a characteristic rotating behavior, which is produced by a bias toward movement on one side that results from the asymmetrical deficit.

The researchers then set about exciting and inhibiting different populations of cells in the STN of the hemiparkinsonian mice, as may happen to these cells during DBS, and measuring the effect of these manipulations on the turning behavior, among other measures of the parkinsonian phenotype. After ruling out local excitatory neurons and glia, they found that high-frequency stimulation of axons in the STN that carry signals from other brain regions improved the mice’s phenotype. Like with DBS in humans, low-frequency stimulation was not effective, and by some measures even worsened the phenotype.

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Further, high-frequency stimulation of a population of cells in the primary motor cortex (M1) from which some of the axons in the STN originated also improved the mice’s symptoms. Given that stimulation of the neurons influenced by these axons could not account for the improvement in phenotype, the researchers suggest that signals could be traveling backwards along the axon from the site of stimulation in the STN, relieving the parkinsonism by shifting activity in M1 away from low frequency bursting.

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Though other mechanisms may still account for the efficacy of STN DBS, this research revealed a surprising mechanism by which DBS could improve Parkinson’s disease in humans, which was not intended at the time that STN DBS was originally developed. In addition, these results show the power of techniques for targeted dissection of neural circuits like optogenetics. Such methods have the potential to help researchers understand diseases and their treatments, and even provide insight in how to better target such treatments to improve their efficacy or reduce side effects.

To learn about Dr. Gradinaru’s current research, attend her talk on Tuesday, February 7th at 4 pm in the Marilyn G. Farquhar Seminar Room of the Center for Neural Circuits and Behavior.

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Jacob Garrett is a first-year graduate student in the UCSD Neurosciences program, whose main interests within neuroscience include modeling and primary sensory systems.

Can the “dynome” succeed where the connectome cannot?

Recently, the big research push in the neurosciences has been to characterize the complete wiring diagram of the brain – to describe the Connectome.  Though a lofty goal that will likely span another decade or more of work, no one doubts the enormous contribution that it will provide to the understanding of brain functioning.  However, even with the connectome in hand, researchers will still be at a loss to explain the generation, coordination, and consequences of dynamic rhythmic processes that characterize so much of the cognitive neuroscience literature.  To bridge this explanatory gap, Dr. Nancy Kopell, a professor of mathematics and statistics at Boston University, has suggested a new large-scale research undertaking aimed at characterizing the dynamic interaction of brain regions – to describe the “Dynome”.

Dr. Kopell’s work mainly focuses on mathematically characterizing the types of dynamics interactions that take place between cells, within networks, and between brain regions, as well as constructing biophysical models of how these dynamics may occur.  Her research, and that of her contemporaries, has demonstrated that dynamic brain rhythms can be involved in phenomena such as network synchronization, attentional gain, and recruiting brain regions per cognitive demands.  For example, the well-known pyramidal interneuronal gamma (PING) mechanism leading to higher frequency gamma rhythms relies on feedback inhibition.  This inhibition leads to a winner-take-all type scenario whereby the most activated cells end up suppressing more weakly activated ones, and potentially serves as a mechanism of focused attention.

Unfortunately, a difficulty that arises, even within a type brain rhythm, is that the generative mechanisms and modulation of rhythms can vary widely by brain region, cortical layer, and even cell type.  Thus, it becomes critical to outline not only the anatomical structure of a region, as connectomics is attempting, but also the cellular physiology, functional synaptic connectivity, and neuromodulatory profiles present.  Fortunately, technologies that have been emerging over the last few years in experimental neuroscience seem well-suited to provide answers to these exact types of questions.  Specifically, high density electrode recordings, optogenetic manipulation, and large scale 3D imaging of neuronal activity have allowed more in-depth analysis of network activity, small circuit functioning, and cell-type specific physiology.  Additionally, new data analytic techniques are providing ways to characterize activity and understand correlations, while data-driven computational models allow researchers to test potential generative mechanisms.

The neuronal heterogeneity involved in implementing these brain rhythms might seem intimidating, but it is likely that it is necessary for the emergence of many cognitive phenomena.  For example, if the same rhythms are being differentially generated in two brain regions, then the similar operating frequency will facilitate stronger interactions, while the dissimilar implementation may mean distinct computations are being performed.  Moreover, the heterogeneity allows these various regions to be differentially regulated by the same neuromodulators, possibly leading to the diverse set of changes that occur across different cognitive states.  Perhaps the most compelling evidence for this is in the effectiveness of deep brain stimulation in treating various mental illnesses.  Cognitive and behavioral abnormalities exhibited by patients with Parkinson’s disease, depression, or obsessive compulsive disorder all experience dramatic improvements simply by a perturbation of the underlying pathological network dynamics.

Importantly, elucidation of the Dynome can and should occur in concert with that of the Connectome.  The highly plastic nature of the brain means that connections constantly change according to the dynamic activity present.  With that in mind, it seems a static Connectome can never completely capture its architecture.  However, this type of broad framework, large-scale synthesis, and open-ended goals may allow neuroscience to continue its rapid progression towards explaining brain functioning.  As data is continually compiled across various levels of brain functioning, the contributions of both connectomics and dynomics will both be needed to move us from genes, to physiology, to network dynamics and interactions, eventually to cognition and pathology.

To hear more about Dr. Kopell’s specific contributions to the dynome, please attend her talk on Tuesday, January 31 at 4pm in the CNCB Marilyn G. Farquhar Seminar Room. To learn more about her lab and for a list of recent publications, please visit her website: http://math.bu.edu/people/nk/

Ryan Golden is a first-year in the Neurosciences Graduate Program, and is currently rotating in Maxim Bazhenov’s lab.  His interests broadly fall under computational and theoretical neuroscience, and is specifically interested in the biophysical implementation and modulation of reinforcement learning.

 

 

Juvenile mouse pheromone inhibits sexual be

With the past few decades bringing exponential growth in the fields of neuroscience and cell biology, we have begun to understand physiology in a way that our predecessors could only hypothesize. We have identified countless proteins, genes, receptors and channels, traced their interactions, manipulated their properties and even developed drugs that can interact to treat diseases. However, there are still many areas that lack understanding of the players involved in some of the most basic biological processes. Stephen Liberles, PhD, is an associate professor in Cell Biology at Harvard Medical School. His research involves internal and external sensory systems, mainly olfaction and pheromone signaling and sensory modalities of the vagus nerve. Here I will discuss the findings from a 2013 Nature paper regarding juvenile mouse pheromone signaling and inhibition of sexual behavior.

Pheromones come in many forms: urinary volatiles, steroid derivatives and proteins in urine, tears and saliva. However, the function of many of these pheromones is unknown. qPCR using cDNA from a range of ages in mice demonstrated stark age-dependent differences in some peptide expression levels in the extraorbital lacrimal glad (LG). Amongst these peptides was exocrine-gland secreting peptide 22 (ESP22) which was produced in juveniles, but not in adults (see attached figure). The expression levels were highest at 2-3 weeks, corresponding to the time just before puberty. The levels were similar in males and females, and was isolated to the LG. RNA in situ hybridization localized ESP22 to a set of acinar lacrimal cells, the cells responsible for releasing contents into tears. Western blot analysis supported that ESP22 was a component of juvenile tears, using mass spectrometry the Liberles lab was able to identify the primary structure of the peptide.

Once ESP22 had been identified in juvenile mice, the sensory perception pathway was examined. Electrophysiological recordings determined that neurons in the vomeronasal organ (VNO) respond to ESP22, and more specifically require the ion channel TRPC2. This was affirmed by juvenile tears activating VNO neurons, while adult tears produced no recordings. Immunohistochemical staining (cFos) was used to trace the neural circuitry. It was demonstrated that upon activation with ESP22 cFos expression was increased in the medial amygdala (MeA) which receives input from the VNO by the accessory olfactory bulb and sends projetions to the hypothalamic areas controlling reproductive responses. In order to further examine the signaling, TRPC2 knockout (KO) mice were used and demonstrated no activation of the amygdala.

Once the signaling pathway was determined, implicating the need for both ESP22 and TRPC2 receptor, behavioral studies were performed in mice lacking ESP22 (C3H strain) and TRPC2 KO mice. While wild type male mice have a mating preference for adult females, they tend to not mount juveniles. However, when ESP22 was not present, the adult mice had an increased time spent mounting juveniles, and decreased latency between mountings. Similarly, the TRPC2 KO mice also spent more time mounting juveniles. When exogenous ESP22 was added to the C3H juvenile mice, the males with TRPC2 stopped the mating behavior.

Taking together this information supports the findings that ESP22 is a pheromone produced by the acinar cells in the lacrimal gland of juvenile mice before puberty. It activates TRPC2 in adult male mice, which acts on the medial amygdala to inhibit mating behavior. Once the mice reach puberty, the production of ESP22 decreases from the females, thus removing the inhibition from the males, and allowing for regular mating behavior.

For more information and publications from the Liberles lab, please visit the website at https://liberles.med.harvard.edu/home

Amy Taylor is a third year MSTP student in the Neuroscience PhD program at University of California San Diego. Her research focus is on the identification and manipulation of EEG biomarkers in schizophrenia and their relation to functional outcomes.

Role of Sox4 and Sox11 in Early Neuronal Differentiation

Corticogenesis is a complex process requiring proliferation of progenitor cells in the ventricular and subventricular zones, neuronal migration, and synaptogenesis. Early in neural development, radial glia divide to produce excitatory neurons of the cortex. Later on in development, radial glia produce intermediate progenitor cells (IPCs) that detach from the surface of the ventricular zone (VZ) to relocate to the subventricular zone (SVZ). IPCs have limited mitotic potential relative to radial glia, and also produce cells that migrate into the cortex. The timing of activation of molecular pathways involved in proliferation and neural differentiation is critical during corticogenesis, and relies on various transcriptional regulators.

The Sox family of transcriptional regulators is known to alter gene expression and determine cell fate, and they partner with other proteins to exert their effects. The SoxC subfamily of transcriptional regulators (Sox4, Sox11, and Sox12) is thought to be functionally identical and expressed in all neuron subtypes. Chen et al. (2015) assessed the expression levels, protein localization, and putative function of both Sox11 and Sox4 in the developing mouse cortex. Sox4 and Sox11 had similar expression levels across cortical development from E10.5-P10 measured by qRT-PCR, peaking between E12.5-E16.5, a time corresponding to early neuronal differentiation.

While expression levels were similar between Sox11 and Sox4 across corticogenesis, immunohistochemical analysis showed distinct protein localizations. Sox11 localized to differentiated neurons, as expected, and also co-localized with Neurogenin1 (Ngn1), a transient marker of early differentiated neurons, at E12.5. Sox4 was localized to both differentiated neurons as well as IPCs in the SVZ. Given the difference in protein expression patterns seen via IHC, Chen et al. examined the role of Sox4 and Sox11 in neuronal differentiation separately. In vitro analysis of differentiated neuron cultures transfected to either overexpress (GOF) or underexpress (LOF) Sox11 was performed. GOF cultures showed increased neuronal polarization and increased neurite length, whereas LOF cultures showed less mature neurons and decreased neurite length. These results support a role of Sox11 in early neuronal differentiation.

So what might Sox11 be doing functionally in these early differentiating neurons? In order to answer this question, Chen et al. used mice with Sox11 coding sequence flanked by loxP sites, and crossed them with a Cre line expressed from the Emx1 promoter, knocking out Sox11 in all cells derived from cortical germinal zones. At early stages of corticogenesis, Sox11 conditional mutants showed reduced immunohistochemical staining for the immature neuronal marker Tuj1. At later stages, mutants showed reduced levels of the deep cortical layer markers Tbr1 and Ctip2. In vivo electroporation to create LOF Sox11 mutants further illustrated that Sox11 promotes the creation of early born neurons by sacrificing apical progenitor cells, as LOF mutants showed an increased proportion of Sox9+ progenitor cells compared to controls.

Sox11 co-localized with Ngn1, a known transcription factor critical for neuronal maturation, and co-immunoprecipitation experiments were performed using E14.5 cortex to assess the ability of Sox11 to bind Ngn1. Sox11 selectively bound Ngn1, suggesting it may be a binding partner with Sox11. Chen et al. also demonstrated that Sox11 could bind regulatory sequences in NeuroD1, an early neuronal differentiation marker in the forebrain regulated by Ngn1, via ChIP analysis. Follow up luciferase assays using an expression vector containing the NeuroD1 promoter linked to luciferase showed that Sox11, when transfected with Ngn1, had a synergistic effect on the activation of the NeuroD1 promoter. These results suggest that Ngn1 and Sox11 act as binding partners to regulate the expression of NeuroD1.

Chen et al. next assessed the function of Sox4 in IPCs in the SVZ. IHC showed that Sox4 co-localized with Ngn2, a transcription factor previously shown to be present in IPCs, as well as Tbr2, another marker of IPCs. In vivo manipulation of Sox4 levels was performed to ascertain the function of Sox4 in IPCs. Cells were transfected with GFP, along with either a Sox4 GOF or Sox4 LOF vector. Cortex was then stained for Tbr2. GOF cortex had significantly more Tbr2+ cells, and LOF cortex had significantly less Tbr2+ cells compared to control cells. At E12.5, conditional Sox4 mutants had a reduction in the number of Tbr2+ IPCs, indicating Sox4 involvement in IPC specification.

Given the co-localization of Sox4 with both Ngn2 and Tbr2, co-immunoprecipitation experiments using E14.5 cortex were performed, showing that Sox4 binds to both Ngn2 and Tbr2. Tbr2 is a known target of Ngn2, and ChIP analysis revealed that Sox4 is able to bind a promoter region on Tbr2. Transactivation experiments showed that Sox4, but not Ngn2, can increase Tbr2 expression, and that cotransfection of Sox4 and Ngn2 produces similar Tbr2 levels to Sox4 alone. These results suggest that Sox4 and Ngn2 may act via distinct molecular pathways to influence Tbr2 levels in IPCs. Luciferase assays showed that Tbr2 cannot increase Tbr2 levels alone, but when cotransfected with Sox4, Tbr2 levels were elevated over Sox4 alone. These results taken together suggest that Ngn2 and Tbr2 are involved in pathways used by Sox4 to induce IPC specification.

Taken together, these results suggest that Sox4 and Sox11 have distinct roles during corticogenesis, and suggest a model by which temporal regulation of corticogenesis may occur (see figure below). To hear more about Dr. Maria Donoghue’s research, please attend her talk on Tuesday, January 17 at 4pm in the CNCB Marilyn G. Farquhar Seminar Room. To learn more about her lab and for a list of recent publications, please visit her lab website: http://www.donoghuelab.org/.

Molly Kwiatkowski is a third year MD/PhD candidate, currently working in the Consortium for Translational Research in Neuropsychopharmacology.

corticogenesis

What can invertebrates tell us about our brains?

With its hundred billion neurons and quadrillion synapses, the human central nervous system(CNS) can seem intractably complex. Fortunately, there is a class of animals whose nervous systems and behaviors are much more easily understood.  Invertebrates, such as sea slugs and worms, have on the order of only hundreds or thousands of neurons and their connections are extremely well stereotyped. This simplicity makes them amenable to experimentation and modeling, and has allowed scientists to understand the structure and function of their neural circuits.

In his review, Allen I. Selverston, Professor Emeritus at UCSD, asks if information gained from the study of invertebrates can be translated to our understanding of the human CNS.  He focuses on a particularly well characterized type of circuit called Central Pattern Generators (CPG).  CPGs are networks of neurons which produce rhythmic outputs in the absence of sensory feedback, and often control simple motor actions such as feeding or swimming. CPGs are not only found in invertebrates but vertebrates as well, where they control certain low level functions.  An example of a CPG is the leech heartbeat network which is shown in the diagram below.

heartbeat

Leech heartbeat neuronal network

The study CPGs using electrical and chemical manipulation of their constituent neurons has led to three primary types of discoveries.  First, it has revealed how a complex array of ion channels contributes to the distinct activity properties of individual neurons. Second, it has shed light on the types of synapses and how they are modulated and third, how circuits produce functional outputs.

Selverston uses these three types of analysis to explain how many different CPGs from the invertebrate world work. Unfortunately, he concludes that there are very few general principles for the design of these circuits that are transferable from model to model. Each CPG has its own evolutionary history that has crafted it into a bespoke circuit for the unique function that it serves. Moreover, the experimental methods used to study CPGs are unlikely to be effective in more complicated vertebrate systems because they cannot be probed with single cell techniques. This means that while the cellular and synapse level data may broadly applicable, the further study of invertebrate CPGs is unlikely to give us much insight into the human CNS.

Selverston’s review can be found here.

Leo Breston is a first year student in the Neuroscience Graduate Program. He is currently rotating in the Navlaka lab.