Drew LJ, Kheirbek MA, Luna VM, Denny CA, Cloidt MA, Wu MV, Jain S, Scharfman HE, Hen R (2015)

Hippocampus

Robust incorporation of new principal cells into preexisting circuitry in the adult mammalian brain is unique to the hippocampal dentate gyrus (DG). We asked if adult-born granule cells (GCs) might act to regulate processing within the DG by modulating the substantially more abundant mature GCs. Optogenetic stimulation of a cohort of young adult-born GCs (0 to 7 weeks post-mitosis) revealed that these cells activate local GABAergic interneurons to evoke strong inhibitory input to mature GCs. Natural manipulation of neurogenesis by aging—to decrease it—and housing in an enriched environment—to increase it—strongly affected the levels of inhibition. We also demonstrated that elevating activity in adult-born GCs in awake behaving animals reduced the overall number of mature GCs activated by exploration. These data suggest that inhibitory modulation of mature GCs may be an important function of adult-born hippocampal neurons.

Tannenholz L, Jimenez JC, Kheirbek MA (2014)

Frontiers in Behavioral Neuroscience

While the hippocampus has been classically studied for its role in learning and memory, there is significant support for a role of the HPC in regulating emotional behavior. Emerging research suggests these functions may be segregated along the dorsoventral axis of the HPC. In addition to this regional heterogeneity, within the HPC, the dentate gyrus is one of two areas in the adult brain where stem cells continuously give rise to new neurons. This process can influence and be modulated by the emotional state of the animal, suggesting that adult neurogenesis within the DG may contribute to psychiatric disorders and cognitive abilities. Yet, the exact mechanism by which these newborn neurons influence behavior remains unknown. Here, we will examine the contribution of hippocampal neurogenesis to the output of the HPC, and suggest that the role of neurogenesis may vary along the DV axis. Next, we will review literature indicating that anatomical connectivity varies along the DV axis of the HPC, and that this underlies the functional segregation along this axis. This analysis will allow us to synthesize novel hypotheses for the differential contribution of the HPC to cognition and mood.

Lovett-Barron M, Kaifosh P, Kheirbek MA, Danielson N, Zaremba JD, Reardon TR, Turi GF, Hen R, Zemelman BV, Losonczy A (2014)

Science

Fear memories guide adaptive behavior in contexts associated with aversive events. The hippocampus forms a neural representation of the context that predicts aversive events. Representations of context incorporate multisensory features of the environment, but must somehow exclude sensory features of the aversive event itself. We investigated this selectivity using cell type–specific imaging and inactivation in hippocampal area CA1 of behaving mice. Aversive stimuli activated CA1 dendrite-targeting interneurons via cholinergic input, leading to inhibition of pyramidal cell distal dendrites receiving aversive sensory excitation from the entorhinal cortex. Inactivating dendrite-targeting interneurons during aversive stimuli increased CA1 pyramidal cell population responses and prevented fear learning. We propose subcortical activation of dendritic inhibition as a mechanism for exclusion of aversive stimuli from hippocampal contextual representations during fear learning.

Kheirbek MA, Hen R (2014)

Scientific American

To keep memories from becoming jumbled, the brain must encode the distinct features of events and situations in a way that allows them to be distinguished from one another—a process called pattern separation. Pattern separation enables us to distinguish dangerous situations from similar ones that pose no risk. People with defects in this ability may be prone to anxiety disorders. The process occurs in one of the two regions of the brain that generate neurons throughout life. These fledgling cells seem to be critical to pattern separation. Interventions that specifically boost the ranks of rookie neurons could provide new ways to regulate mood and possibly treat conditions such as post-traumatic stress disorder.

Denny CA, Kheirbek MA, Alba EL, Tanaka, KF, Brachman RA, Laughman KB, Tomm NK, Turi GF, Losonczy A, Hen R (2014)

Neuron

Memory traces are believed to be ensembles of cells used to store memories. To visualize memory traces, we created a transgenic line that allows for the comparison between cells activated during encoding and expression of a memory. Mice re-exposed to a fearinducing context froze more and had a greater percentage of reactivated cells in the dentate gyrus (DG) and CA3 than mice exposed to a novel context. Over time, these differences disappeared, in keeping with the observation that memories become generalized. Optogenetically silencing DG or CA3 cells that were recruited during encoding of a fear-inducing context prevented expression of the corresponding memory. Mice with reduced neurogenesis displayed less contextual memory and less reactivation in CA3 but, surprisingly, normal reactivation in the DG. These studies suggest that distinct memory traces are located in the DG and in CA3 but that the strength of the memory is related to reactivation in CA3.

Kheirbek MA, Hen R (2013)

Cell

Fifteen years ago, the generation of new neurons in adulthood was documented in the human hippocampus, but lingering questions have remained about the extent of this process. In this issue of Cell, Spalding et al. provide elegant evidence for continued neurogenesis into adulthood at rates that suggest it may play a significant role in human behavior.

Ahmari SE, Spellman T, Douglass N, Kheirbek MA, Deisseroth K, Gordon JA, Hen R (2013)

Science

Although cortico-striato-thalamo-cortical (CSTC) circuit dysregulation is correlated with obsessive compulsive disorder (OCD), causation cannot be tested in humans. We used optogenetics in mice to simulate CSTC hyperactivation observed in OCD patients. Whereas acute orbitofrontal cortex (OFC)–ventromedial striatum (VMS) stimulation did not produce repetitive behaviors, repeated hyperactivation over multiple days generated a progressive increase in grooming, a mouse behavior related to OCD. Increased grooming persisted for 2 weeks after stimulation cessation. The grooming increase was temporally coupled with a progressive increase in light-evoked firing of postsynaptic VMS cells. Both increased grooming and evoked firing were reversed by chronic fluoxetine, a first-line OCD treatment. Brief but repeated episodes of abnormal circuit activity may thus set the stage for the development of persistent psychopathology.

Kheirbek MA, Drew LJ, Costantini DO, Burghardt N, Tannenholz L, Ahmari SE, Zeng H, Fenton AA, Hen R (2013)

Neuron

The dentate gyrus (DG), in addition to its role in learning and memory, is increasingly implicated in the pathophysiology of anxiety disorders. Here, we show that, dependent on their position along the dorsoventral axis of the hippocampus, DG granule cells (GCs) control specific features of anxiety and contextual learning. Using optogenetic techniques to either elevate or decrease GC activity, we demonstrate that GCs in the dorsal DG control exploratory drive and encoding, not retrieval, of contextual fear memories. In contrast, elevating the activity of GCs in the ventral DG has no effect on contextual learning but powerfully suppresses innate anxiety. These results suggest that strategies aimed at modulating the excitability of the ventral DG may be beneficial for the treatment of anxiety disorders.

Preview by Fournier and Duman (pdf)

Kheirbek MA, Klemenhagen KK, Sahay A, Hen R (2012)

Nature Neuroscience

Although an influence of adult neurogenesis in mediating some of the effects of antidepressants has received considerable attention in recent years, much less is known about how alterations in this form of plasticity may contribute to psychiatric disorders such as anxiety and depression. One way to begin to address this question is to link the functions of adult-born hippocampal neurons with specific endophenotypes of these disorders. Recent studies have implicated adult-born hippocampal neurons in pattern separation, a process by which similar experiences or events are transformed into discrete, non-overlapping representations. Here we propose that impaired pattern separation underlies the overgeneralization often seen in anxiety disorders, specifically post-traumatic stress disorder and panic disorder, and therefore represents an endophenotype for these disorders. The development of new, pro-neurogenic compounds may therefore have therapeutic potential for patients who display pattern separation deficits.

Kheirbek MA, Tannenholz L, Hen R (2012)

Journal of Neuroscience

Adult-generated granule cells (GCs) in the dentate gyrus (DG) exhibit a period of heightened plasticity 4 – 6 weeks postmitosis. However, the functional contribution of this critical window of plasticity to hippocampal neurogenesis and behavior remains unknown. Here, we show that deletion of NR2B-containing NMDA receptors from adult-born GCs impairs a neurogenesis-dependent form of LTP in the DG and reduces dendritic complexity of adult-born GCs, but does not impact their survival. Mice in which the NR2B-containing NMDA receptor was deletedfrom adult-born GCs did not differfrom controlsin baseline anxiety-like behavior or discrimination of very different contexts, but were impaired in discrimination of highly similar contexts. These results indicate that NR2B-dependent plasticity of adult-born GCs is necessary for fine contextual discrimination and is consistent with their proposed role in pattern separation.

Kheirbek MA, Hen R (2011)

Neuropsychopharmacology

Sahay A, Scobie KN, Hill SH, O’Carroll CM, Kheirbek MA, Burghardt N, Fenton AA, Dranovsky A, Hen R (2011)

Nature

Adult hippocampal neurogenesis is a unique form of neural circuit plasticity that results in the generation of new neurons in the dentate gyrus throughout life. Neurons that arise in adults (adult-born neurons) show heightened synaptic plasticity during their maturation and can account for up to ten percent of the entire granule cell population. Moreover, levels of adult hippocampal neurogenesis are increased by interventions that are associated with beneficial effects on cognition and mood, such as learning, environmental enrichment, exercise and chronic treatment with antidepressants. Together, these properties of adult neurogenesis indicate that this process could be harnessed to improve hippocampal functions. However, despite a substantial number of studies demonstrating that adult-born neurons are necessary for mediating specific cognitive functions, as well as some of the behavioural effects of antidepressants, it is unknown whether an increase in adult hippocampal neurogenesis is sufficient to improve cognition and mood. Here we show that inducible genetic expansion of the population of adult-born neurons through enhancing their survival improves performance in a specific cognitive task in which two similar contexts need to be distinguished. Mice with increased adult hippocampal neurogenesis show normal object recognition, spatial learning, contextual fear conditioning and extinction learning but are more efficient in differentiating between overlapping contextual representations, which is indicative of enhanced pattern separation. Furthermore, stimulation of adult hippocampal neurogenesis, when combined with an intervention such as voluntary exercise, produces a robust increase in exploratory behaviour. However, increasing adult hippocampal neurogenesis alone does not produce a behavioural response like that induced by anxiolytic agents or antidepressants. Together, our findings suggest that strategies that are designed to increase adult hippocampal neurogenesis specifically, by targeting the cell death of adult-born neurons or by other mechanisms, may have therapeutic potential for reversing impairments in pattern separation and dentate gyrus dysfunction such as those seen during normal ageing.

Beeler JA, Cao ZFH, Kheirbek MA, Ding Y, Koranda J, Murakami M, Zhuang X (2010)

Annals of Neurology

Objective: Dopamine (DA) is critical for motor performance, motor learning, and corticostriatal plasticity. The relationship between motor performance and learning, and the role of DA in the mediation of them, however, remain unclear.
Methods: To examine this question, we took advantage of PITx3-deficient mice (aphakia mice), in which DA in the dorsal striatum is reduced by 90%. PITx3-deficient mice do not display obvious motor deficits in their home cage, but are impaired in motor tasks that require new motor skills. We used the accelerating rotarod as a motor learning task.
Results: We show that the deficiency in motor skill learning in PITx3(-/-) is dramatic and can be rescued with levodopa treatment. In addition, cessation of levodopa treatment after acquisition of the motor skill does not result in an immediate drop in performance. Instead, there is a gradual decline of performance that lasts for a few days, which is not related to levodopa pharmacokinetics. We show that this gradual decline is dependent on the retesting experience.
Interpretation: This observation resembles the long-duration response to levodopa therapy in its slow buildup of improvement after the initiation of therapy and gradual degradation. We hypothesize that motor learning may play a significant, underappreciated role in the symptomatology of Parkinson disease as well as in the therapeutic effects of levodopa. We suggest that the important, yet enigmatic long-duration response to chronic levodopa treatment is a manifestation of rescued motor learning.

Kheirbek MA, Beeler JA, Chi W, Zhuang X (2010) Learning and Memory

In appetitive Pavlovian learning, animals learn to associate discrete cues or environmental contexts with rewarding outcomes, and these cues and/or contexts can potentiate an ongoing instrumental response for reward. Although anatomical substrates underlying cued and contextual learning have been proposed, it remains unknown whether specific molecular signaling pathways within the striatum underlie one form of learning or the other. Here, we show that while the striatum-enriched isoform of adenylyl cyclase (AC5) is required for cued appetitive Pavlovian learning, it is not required for contextual appetitive learning. Mice lacking AC5 (AC5KO) could not learn an appetitive Pavlovian learning task in which a discrete signal light predicted reward delivery, yet they could form associations between context and either natural or drug reward, which could in turn elicit Pavlovian approach behavior. However, unlike wild-type (WT) mice, AC5KO mice could not use these Pavlovian conditioned stimuli to potentiate ongoing instrumental behavior in a Pavlovian-to-instrumental transfer paradigm. These data suggest that AC5 is specifically required for learning associations between discrete cues and outcomes in which the temporal relationship between conditioned stimulus (CS) and unconditioned stimulus (US) is essential, while alternative signaling mechanisms may underlie the formation of associations between context and reward. In addition, loss of AC5 compromises the ability of both contextual and discrete cues to modulate instrumental behavior.

Beeler JA, Cao ZFH, Kheirbek MA, Zhuang X (2009)

Neuropsychopharmacology

Both the dorsal and ventral striatum have been demonstrated to have a critical role in reinforcement learning and addiction. Dissecting the specific function of these striatal compartments and their associated nigrostriatal and mesoaccumbens dopamine pathways, however, has proved difficult. Previous studies using lesions to isolate the contribution of nigrostriatal and mesoaccumbens dopamine in mediating the locomotor and reinforcing effects of psychostimulant drugs have yielded inconsistent and inconclusive results. Using a naturally occurring mutant mouse line, aphakia, that lacks a nigrostriatal dopamine pathway but retains an intact mesoaccumbens pathway, we show that the locomotor activating effects of cocaine, including locomotor sensitization, are dependent on an intact nigrostriatal dopamine projection. In contrast, cocaine reinforcement, as measured by conditioned place preference and cocaine sensitization of sucrose preference, is intact in these mice. In light of the well-established role of the nucleus accumbens in mediating the effects of psychostimulants, these data suggest that the nigrostriatal pathway can act as a critical effector mechanism for the nucleus accumbens highlighting the importance of intrastriatal connectivity and providing insight into the functional architecture of the striatum.

Kheirbek MA, Britt JP, Beeler JA, Ishikawa Y, McGehee DS, Zhuang X (2009)

Journal of Neuroscience

Dopamine (DA)-dependent corticostriatal plasticity isthoughtto underlie incremental procedural learning. A primary effector of striatal DA signaling is cAMP, yet its role in corticostriatal plasticity and striatum-dependent learning remains unclear. Here, we show that genetic deletion of a striatum-enriched isoform of adenylyl cyclase, AC5 knock-out (AC5KO), impairs two forms of striatum-dependent learning and corticostriatal synaptic plasticity. AC5KO mice were severely impaired in acquisition of a response strategy in the cross maze, a striatum-dependent task requiring a correct body turn to find a goal arm. In addition, AC5KO mice were impaired in acquisition of a motor skill, as assessed by the accelerated rotarod. Slice electrophysiology revealed a deficit in corticostriatal long-term depression (LTD) after high-frequency stimulation oftissuefrom AC5KOmice. LTD was rescued by activation of either presynaptic cannabinoidtype 1 (CB1 ) receptors or postsynaptic metabotropic glutamate receptors (mGluRs), suggesting a postsynaptic role of AC5– cAMP, upstream of endocannabinoid release. In striatopallidal-projecting medium spiny neurons, DA D2 receptors are negatively coupled to cAMP production, and activation of these receptors is required for endocannabinoid release and corticostriatal LTD. Recordings from striato pallidal neurons indicated that this is mediated by AC5, because coactivation of D2 and mGluRs could induce LTD in wild-type but not in AC5KO neurons. To further examine the role of cAMP in corticostriatal plasticity, we elevated cAMP in striatal neurons of wild-type mice via the recording electrode. Under these conditions, corticostriatal LTD was eliminated. Together, these data suggest an AC5– cAMP– endocannabinoid–CB1 signaling pathway in corticostriatal plasticity and striatum-dependent learning.

Kheirbek MA, Beeler JA, Ishikawa Y, Zhuang X (2008)

Journal of Neuroscience

In associative learning, animals learn to associate external cues or their own actions with appetitive or aversive outcomes. Although the dopamine (DA) system and the striatum/nucleus accumbens have been implicated in both the pavlovian and instrumental form of associative learning, whether specific neuronal signaling mechanisms underlie oneform orthe other is unknown.Here, we reportthatthe striatum-enriched isoform of adenylyl cyclase (AC), AC5, is selectively required for appetitive pavlovian learning. Mice with genetic deletion of AC5 (AC5KO) acquired instrumental responding yet were unable to use cues that predicted reward delivery. The specificity of this deficit was confirmed by an inability of AC5KO mice to learn a simple appetitive pavlovian conditioning task. Conversely, AC5KO mice showed intact aversive pavlovian learning, suggesting the deficit was specific for learning about appetitive outcomes. Our results suggest that AC5 is a critical component of DA-dependent strengthening of stimulus–reward contingencies.

Kheirbek MA (2007)

Journal of Neuroscience

Hale ME, Kheirbek MA, Schriefer JE, Prince VE (2004)

Journal of Neuroscience

Hox genes are critical for establishing the segmental pattern of the vertebrate hindbrain. Changes in their expression can alter neural organization of hindbrain segments and may be a mechanism for brain evolution. To test the hypothesis that neurons induced through changes inHox gene expression can integrate into functional neural circuits, we examined the roles of ectopic Mauthner cells (M-cells) in the escape response of larval zebrafish. The activity of the paired Mauthner cells in rhombomere 4 (r4) has been shown to be critical for generating a high-performance startle behavior in response to stimulation of the tail (Liu and Fetcho, 1999). Previous studies have found that misexpression of particular Hox genes causes ectopic M-cells to be generated in r2 in addition to the r4 cells (Alexandre et al., 1996; McClintock et al., 2001). With calcium imaging, we found that the homeotically transformed neurons respond to startle stimuli. To determine the roles of ectopic and endogenous M-cells in the behavior, we lesioned the r2, r4, or both M-cells with cell-specific laser lesion and examined the effect on startle performance. Lesion of the normal M-cells did not decrease escape performance when the ectopic cells were present. These results indicate that the homeotically transformed Mauthner cells are fully functional in the escape circuit and are functionally redundant with normal M-cells. We suggest that such functional redundancy between neurons may provide a substrate for evolution of neural circuits.

McClintock JM, Kheirbek MA, Prince VE (2002)

Development

We have used a morpholino-based knockdown approach to investigate the functions of a pair of zebrafish Hox gene duplicates, hoxb1a and hoxb1b, which are expressed during development of the hindbrain. We find that the zebrafish hoxb1 duplicates have equivalent functions to mouse Hoxb1 and its paralogue Hoxa1. Thus, we have revealed a ‘function shuffling’ among genes of paralogue group 1 during the evolution of vertebrates. Like mouse Hoxb1, zebrafish hoxb1a is required for migration of the VIIth cranial nerve branchiomotor neurons from their point of origin in hindbrain rhombomere 4 towards the posterior. By contrast, zebrafish hoxb1b, like mouse Hoxa1, is required for proper segmental organization of rhombomere 4 and the posterior hindbrain. Double knockdown experiments demonstrate that the zebrafish hoxb1 duplicates have partially redundant functions. However, using an RNA rescue approach, we reveal that these duplicated genes do not have interchangeable biochemical functions: only hoxb1a can properly pattern the VIIth cranial nerve. Despite this difference in protein function, we provide evidence that the hoxb1 duplicate genes were initially maintained in the genome because of complementary degenerative mutations in defined cis-regulatory elements.