# **MECHANISMS OF NEURAL CIRCUIT FORMATION**

**Topic Editors Joshua A. Weiner, Robert W. Burgess and James Jontes**

MOLECULAR NEUROSCIENCE

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**ISSN** 1664-8714 **ISBN** 978-2-88919-403-2 **DOI** 10.3389/978-2-88919-403-2

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## **MECHANISMS OF NEURAL CIRCUIT FORMATION**

Topic Editors: **Joshua A. Weiner,** The University of Iowa, USA **Robert W. Burgess,** The Jackson Laboratory, USA **James Jontes,** Ohio State University, USA

False-colored montage of fluorescently-labeled pyramidal neurons in the cortex of a Thy1-YFPH transgenic mouse. Image provided by Drs. Andrew Garrett and Joshua Weiner, taken from work reviewed in Weiner and Jontes (2013), part of this e-book.

# Table of Contents

*139 Synaptic Clustering During Development and Learning: The why, when, and how*

Johan Winnubst and Christian Lohmann

Masahito Yamagata and Joshua R. Sanes

## Introduction to mechanisms of neural circuit formation

*Joshua A. Weiner 1, James D. Jontes 2 and Robert W. Burgess 3 \**

*1 Department of Biology, University of Iowa, Iowa City, IA, USA*

*2 Department of Neuroscience, The Ohio State University, Columbus, OH, USA*

*3 The Jackson Laboratory, Bar Harbor, ME, USA*

*\*Correspondence<EMAIL_ADDRESS>
#### *Edited by:*

*Robert J. Harvey, UCL School of Pharmacy, UK*

#### *Reviewed by:*

*Robert J. Harvey, UCL School of Pharmacy, UK*

Much progress has been made in recent years toward understanding the underlying causes of neurodevelopmental disorders. Whereas catastrophic failures in early events such as cell fate specification or cell migration give rise to profound developmental defects including microcephaly and lissencephaly, more subtle conditions such as autism, intellectual disability or neuropsychiatric disorders increasingly appear to result from comparatively minor changes in neural circuit formation and function. The formation of proper neuronal circuitry relies on later developmental processes such as axon guidance, the arborization both of axons and their target dendrites, the recognition of appropriate synaptic partners, the establishment and maturation of synaptic connections, and the subsequent elimination of improper connections. The research topic presented here, "Mechanisms of Neural Circuit Formation," addresses recent advances in our understanding of the cellular and molecular bases of these processes.

The papers in this volume generally fall into three broad areas of developmental neurobiology, including new techniques for the study of these processes: (1) cell adhesion molecules (CAMs) and their downstream roles in cell identity, recognition, and synaptic specificity; (2) axon guidance, formation of terminals, and dendritic arborization; and (3) formation of synaptic structures themselves, the essential final step in circuit formation which, nevertheless, remains subject to remodeling and plasticity throughout development and even in adult animals.

The volume opens with a review by Sokolowski and Corbin (2012), highlighting the importance of neuronal circuit formation for behavior through an examination of the development of the limbic circuitry. This is followed by a series of papers on CAMs: a review from Weiner and Jontes (2013), and a hypothesis paper from Yagi (2012) that describe the roles of protocadherins. These are followed by the new results of Prasad and Weiner (2011) on a requirement for γ-protocadherins in Ia afferent terminal formation in the spinal cord. Next is a review from Garrett et al. (2012) on the immunoglobulin (Ig) superfamily proteins Down Syndrome Cell Adhesion Molecule and related proteins (DSCAMs), addressing issues of molecular diversity, cell identity and cell adhesion as they contribute to synapse and circuit formation. Finally, Enriquez-Barreto et al. (2012) present original research on the function of the Ig superfamily member Neural Cell Adhesion Molecule (NCAM) in axon pathfinding and organization in the thalamocortical system.

This last paper transitions into a section focused on axon guidance, including both signaling and cytoskeletal mechanisms. Sakai and Kaprielian (2012) review the literature on the guidance of longitudinally projecting axons, a prominent focus of study in both vertebrates and invertebrates. An original paper by Leslie et al. (2012), examines the requirement of RhoA in sensory axon guidance. The relationship of axon guidance mechanisms to other processes involved in synaptic specificity is highlighted in a review of semaphorin signaling by Yoshida (2012). Finally, the importance of FGF22 in axon termination and synaptogenesis in the lateral geniculate is demonstrated by Singh et al.'s original research (2012).

The penultimate group of papers concerns mechanisms regulating the formation of pre-and post-synaptic structures. The assembly and plasticity of the presynaptic active zone is reviewed in Clarke et al. (2012). A review from Winnubst and Lohmann (2012) discusses the mechanisms and implications for synaptic clustering on target neurons. An original research paper from Murphy et al. (2012) investigates the developmental regulation of AMPA receptor trafficking proteins.

The E-book closes with two papers describing powerful new techniques for studying neural circuit formation that are revealing novel biology both in Drosophila, as described by Nose (2012), and in mice, as described in Yamagata and Sanes (2012).

Together, these papers provide a broad reflection of the state of our knowledge concerning the molecular cues that direct axon and dendrite development, promote the formation of synaptic connections, and allow the refinement of connectivity and plasticity during development. An interesting observation that comes from these studies is that few, if any, of the key molecules are single-purpose; each pathway is used at distinct stages of neural development and serves multiple functions. The extent to which these functions relate varies. For example, semaphorins broadly influence both axonal and dendritic development, perhaps not surprisingly given the large number of different ligands and receptors involved. Fibroblast Growth Factor family members (FGFs), critical players in a wide variety of developmental events in the early embryo, are much later re-purposed to regulate the formation of synaptic circuitry. CAMs like the γ-protocadherins and DSCAMs, regulate several key processes, including neuronal survival, dendrite arborization, and axonal targeting; here, it remains unclear whether these functions are achieved through common signaling pathways, or whether they represent distinct roles for these CAMs. Thus, despite the daunting complexity of the task, the cast of key players is not increasing as rapidly as the number of roles assigned to each cast member. This could facilitate future progress by focusing efforts on a smaller number of molecules. Conversely, efforts could be complicated by the need to dissect primary and secondary effects for each pathway. This highlights the need to be particularly rigorous in defining where, when and how the functions of these proteins are analyzed. As the methods and specificity of these analyses improve, so will our understanding of neural circuit formation.

Considering the finely-tuned complexity of the mature nervous system, the robustness of neurodevelopment is amazing; despite genetic and environmental variability during

## **REFERENCES**

development, and the stochastic variation inherent in biological systems, most brains end up working pretty well. The experimental investigation of small circuits and defined aspects of neural circuit formation is facilitating progress in this area, as the papers in this volume demonstrate. However, a key challenge for the future is to integrate the information we are gaining on individual molecular pathways into a larger model in order to understand how the neural circuitry of the mature nervous system is assembled during development.

of retinal nerve terminals in the dorsal lateral geniculate nucleus. *Front. Mol. Neurosci.* 4:61. doi: 10.3389/fnmol.2011.00061

identifying synaptic connections in mice by fluorescence complementation (GRASP). *Front. Mol. Neurosci.* 5:18. doi: 10.3389/fnmol.2012.00018

Yoshida, Y. (2012). Semaphorin signaling in vertebrate neural circuit assembly. *Front. Mol. Neurosci.* 5:71. doi: 10.3389/fnmol.2012.00071

*Received: 11 April 2013; accepted: 26 April 2013; published online: 13 May 2013.*

*Citation: Weiner JA, Jontes JD and Burgess RW (2013) Introduction to mechanisms of neural circuit formation. Front. Mol. Neurosci. 6:12. doi: 10.3389/ fnmol.2013.00012*

*Copyright © 2013 Weiner, Jontes and Burgess. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

## Wired for behaviors: from development to function of innate limbic system circuitry

## *Katie Sokolowski and Joshua G. Corbin\**

*Children's National Medical Center, Center for Neuroscience Research, Children's Research Institute, Washington, DC, USA*

#### *Edited by:*

*Robert W. Burgess, The Jackson Laboratory, USA*

#### *Reviewed by:*

*Erik Maronde, University of Frankfurt, Germany Hansen Wang, University of Toronto, Canada*

#### *\*Correspondence:*

*Joshua G. Corbin, Children's National Medical Center, Center for Neuroscience Research (CNR), Children's Research Institute, 111 Michigan Avenue, NW, Washington, DC 20010-2970, USA. e-mail<EMAIL_ADDRESS>
The limbic system of the brain regulates a number of behaviors that are essential for the survival of all vertebrate species including humans. The limbic system predominantly controls appropriate responses to stimuli with social, emotional, or motivational salience, which includes innate behaviors such as mating, aggression, and defense. Activation of circuits regulating these innate behaviors begins in the periphery with sensory stimulation (primarily via the olfactory system in rodents), and is then processed in the brain by a set of delineated structures that primarily includes the amygdala and hypothalamus. While the basic neuroanatomy of these connections is well-established,much remains unknown about how information is processed within innate circuits and how genetic hierarchies regulate development and function of these circuits. Utilizing innovative technologies including channel rhodopsin-based circuit manipulation and genetic manipulation in rodents, recent studies have begun to answer these central questions. In this article we review the current understanding of how limbic circuits regulate sexually dimorphic behaviors and how these circuits are established and shaped during pre- and post-natal development. We also discuss how understanding developmental processes of innate circuit formation may inform behavioral alterations observed in neurodevelopmental disorders, such as autism spectrum disorders, which are characterized by limbic system dysfunction.

**Keywords: innate, limbic system, development, olfaction, amygdala, hypothalamus, behaviors**

## **INTRODUCTION**

The limbic system links external cues possessing emotional, social, or motivational relevance to a specified set of contextual and species-specific appropriate behavioral outputs. While a fair amount of these behaviors are enhanced through experiential learning and reinforcement, a number of these behaviors are innate or inborn, meaning that they manifest without prior learning. These innate behaviors include courtship, maternal care, defense (both to conspecific and predator cues) and establishment of social hierarchy, all of which ensure survival of the individual or offspring and propagation of the species. These behaviors are regulated and influenced by sensory stimuli such as touch, sound, and, most importantly in rodents, smell. An animal's inability to correctly detect or process social or environmental cues results in abnormal social behaviors and increases risk of attack and/or predation. In humans, abnormal development of aspects of innate behavior, most prominently circuits that regulate social behavior, appear to underlie disorders such as autism spectrum disorders and schizophrenia that are characterized by inappropriate or altered social interactions.

Until relatively recently, humans were the only species thought to possess emotion. Initially documented by Papez (1937) and elaborated by MacLean (1949), social cognition occurs through a complex neural network of interconnected structures, which includes areas in the ventromedial aspect of the temporal and frontal lobes, and their connections with the hypothalamus and brainstem. This neural network, dubbed the "limbic system" is centered around the amygdala, a small almond shaped structure located deep within the temporal lobe. Emotional salience, produced in the amygdala, is generally thought of as a prime driving force behind innate human behaviors, typically social in nature (Brothers, 1989; Barbas, 1995; Aggleton, 2000; LeDoux, 2012). As the scientific community accepted emotions such as fear, anxiety, reward, and attraction as a result of neural wiring in humans, other species including rodents were gradually accepted as possessing similar circuits and, therefore, similar emotions (see **Figure 1** for comparison of human and rodent limbic system structures). Since the realization that emotions are not exclusively human, understanding the neural circuits involved in processing emotions and other social cues has advanced rapidly through the use of experimental rodent models. In rodent models, emotional states (e.g., fear, anxiety, and social receptivity) are generally quantified by their behaviors. When translating from rodent models to humans, it is important to understand that the sensory inputs of rodents are primarily olfactory, auditory, and somatosensory, with minimal visual inputs. Therefore, in this review we focus primarily on chemosensation in the rodent and how it relates to innate limbic responses to social conspecific cues such as mating, maternal care, and territorial behaviors as well as non-social defensive responses to predator cues.

## **NEUROANATOMY OF INNATE BEHAVIORS**

Most of our knowledge of the circuitry that regulates innate behaviors has come from structural or cellular loss-of-function lesion and cytotoxic injury approaches. However, as the collection of brain regions within the innate circuitry contains a number of

(AOB, red). Together these structures facilitate the execution and reinforcement of innate behaviors.

intertwined fibers of passage, lesion studies by their very nature are limited in their ability to discern the function of discrete nuclei from other connected brain regions. Despite this drawback, these types of classical studies have painted a relatively consistent picture of the major structures that comprise innate circuitry. These structures include the main and accessory olfactory system, olfactory/piriform cortex, amygdala, bed nucleus of stria terminalis (BNST) and hypothalamus (Swanson, 2000; Dulac and Wagner, 2006) (see **Table 1** for abbreviations).

Many behaviors such as fear/aversion to predator odors and reward/attraction to odors of the opposite sex are considered to be innate, meaning no prior learning is needed for their manifestation. For example, a naïve female rodent shows preference to male urine odors over female or no odors (Drickamer, 1992; Sawrey and Dewsbury, 1994). Similarly, a laboratory rat or mouse that has never encountered a predator of any kind will display stereotypical signs of fear and avoidance in response to predator odors (Apfelbach et al., 2005). Specific fear responses

are also initiated by the detection of alarm pheromones thought to be emitted from dead or stressed conspecifics. These alarm pheromones are detected in the Grueneberg Ganglion, located in the tip of the rodent nose (Brechbühl et al., 2008). With the exception of alarm pheromones, innate responses have been tied to specific chemicals (Papes et al., 2010; Ferrero et al., 2011; Isogai et al., 2011) that are detected by two organs in the nose: the vomeronasal organ (VNO) and to a lesser extent the main olfactory epithelium (MOE). The VNO, located in the palate, primarily detects non-volatile chemicals such as pheromones with high specificity, while the MOE located on turbinates deep in the nasal cavity, detects volatile chemicals. Sensory input from the VNO and MOE are received by and processed in the accessory olfactory bulb (AOB) and main olfactory bulb (MOB), respectively. Projections from the AOB and MOB directly or indirectly synapse on a number of higher order structures including the olfactory/piriform cortex and amygdala. The amygdala is generally believed to be a central processing station where the level of salience is imparted to a given stimulus (or stimuli) (LeDoux, 1993). The amygdala then sends projections to the hypothalamus for further integration and coordination with the brain stem to initiate the body's "fight or flight" responses (e.g., increase in blood pressure, respiratory rate, etc.) (Swanson and Petrovich, 1998). Although we will focus our attention on the VNO-AOBamygdalar-BNST-hypothalamic circuit (see **Figure 2**), the main components of the innate circuit, we would like to emphasize that these brain hubs and their many feedback loops are not the sole components of a highly complex neural network important for the regulation of sociability and innate emotions. We begin by summarizing what is currently known regarding the neuroanatomy of circuits for olfactory-based reproductive, maternal care, predator defense and conspecific defense (aggression) rodent innate behaviors and the individual functions of these nuclei in information processing.

## **MATING BEHAVIORS**

Mating behaviors in males and females consist of two phases: the initial appetitive phase followed by the consummatory phase. In males the appetitive phase includes angiogenital chemoinvestigation, or sniffing, of the female. Pheromonal stimulation of the VNO-AOB olfactory system is relayed to the medial amygdala (MeA), usually via direct connections (Meurisse et al., 2009; Kang et al., 2011). The MeA acts as a hub, dispersing the signal to the BNST, and to anatomically segregated subsets of nuclei of the hypothalamus including the medial preoptic nucleus (mPN), ventrolateral portion of the ventromedial hypothalamus (VMHvl) and ventral premammillary nucleus (PMNv) (Emery and Sachs, 1976). Lesion studies have found the mPN of the hypothalamus to be intimately tied to female preference and pursuit (Kondo and Arai, 1995; Been and Petrulis, 2010). The mPN integrates inputs from the MeA either directly or via the BNST to increase dopamine levels (Newman, 1999; Hull and Dominguez, 2006; Balthazart and Ball, 2007). The mPN then signals to the ventral tegmental area (VTA) and nucleus accumbens (NuAc) to initiate appetitive phase responses such as sniffing. The same circuit (VNO-AOB-MeA-BNST-mPN) also controls consummatory phase behaviors such as mounting, intromission and

### **Table 1 | Abbreviations of limbic structures and summary of their role in innate behaviors.**

ejaculation via afferents to VMHvl and then areas of the midbrain and spinal cord: periaqueductal gray (PAG), nucleus paragiganta and finally the lumbosacral spinal cord (see **Figure 3A**) (Marson, 2004; Normandin and Murphy, 2011a,b).

Female innate reproductive behaviors can be initiated through the same olfactory-amygdala circuit as males. Both the appetitive phase and consummatory phase of female mating begins with pheromonal cues picked up by the VNO and MOE (Baum and Kelliher, 2009). Signals are then passed to MeA via the olfactory bulb (Kang et al., 2011). Afferents from the MeA connect to the mPN of the hypothalamus directly or via the BNST and PMNv in a similar fashion as in the male circuit. While the mPN controls both the appetitive and consummatory phase in males, the female mPN primarily influences appetitive responses such as approaching a male to mate or proceptive behaviors (ear twitching, running short distances away—"teasing"). However the mPN is upstream of female consummatory behaviors, explicitly lordosis, which is initiated in the VMHvl. Lesioning of the VMH results in a decrease in lordosis while electrical stimulation of this region produces lordosis in primed females out of context (no male present) (Pfaff and Sakuma, 1979).  Further supporting these lesioning studies, many of these regions also show increased expression of the activity-dependent intermediate early gene, cFos, after sexual behavior (Coolen et al., 1996). Female consummatory behaviors such as lordosis, similar to males, are also relayed to the PAG, nucleus paragiganta and the lumbosacral spinal cord (Lonstein and Stern, 1998).

Quite interestingly, disruption of particular portions of the above-mentioned reproductive circuit results in male behaviors in females or otherwise altered sexual behaviors. Specifically, surgical removal of the VNO or genetic deletion of *TRPC2*, a channel involved in translating pheromone reception into an electrical signal in olfactory receptor neurons, has been shown to increase male mounting behaviors toward other males (Leypold et al., 2002). Conversely, female mice without a functioning VNO (*TRPC*2−*/*− females) mount males (Leypold et al., 2002; Stowers et al., 2002). Moreover, in V1R receptor knockout (V1R receptors in the VNO identify physiological state of the animal) male mice display a decrease in mounts with females, and females display decreased maternal aggression (Del Punta et al., 2002). Despite these interesting findings, results to the contrary have been observed in studies directly probing the role of VNO in sex discrimination in mice and other rodents using either volatile (detected in MOE) or non-volatile (detected in the VNO) urinary

**FIGURE 2 | Limbic processing of olfactory information in the rodent.** The rodent limbic system is highly influenced by olfactory cues received by the main olfactory epithelium (MOE, purple) and vomeronasal organ (VNO, red). The Grueneberg ganglion (pink), which senses stress in conspecifics, is depicted in the tip of the rodent nose. The VNO, located on the palate, of the mouth detects non-volatile or lipophilic chemicals that are channeled by the tongue through a pore in the roof of the mouth. Volatile chemical scents are more readily aerosolized and travel further back into the nasal cavity to reach the MOE on the turbinates. Projections from sensory neurons in the VNO and MOE are received in the accessory olfactory bulb (AOB) and main olfactory bulb (MOB), respectively, located in the brain. Signal is then passed to other structures of the limbic system including the amygdala (green), bed nucleus of stria terminalis (BNST, blue), and hypothalamus (Hypo, yellow).

odors as a stimulus (Beauchamp et al., 1982; Petrulis et al., 1999; Pankevich et al., 2004). When exposed to whole urine, mice with their VNO removed compensated by using their MOE to detect volatile discriminatory odors. Yet, mice lacking a VNO lose their discriminatory abilities when exposed exclusively to non-volatile odor elements of urine undetectable by the MOE (Keller et al., 2006). While these results reveal a partially redundant role for the MOE in sex discrimination, it appears clear that the VNO is central to the expression of appropriate sex-specific mating behaviors. Interestingly, other accounts of unusual feminization also occur by lesioning deeper portions of the male innate reproductive circuit. Lordosis, a female consummatory behavior, has been observed in males after lesioning the preoptic nucleus of the hypothalamus (Hennessey et al., 1986). Thus, appropriate sexual behavior appears to be controlled at multiple levels of the circuit, from pheromone detection in the VNO down to the hypothalamus and spinal cord.

#### **DEFENSE/FEAR**

Innate fear and the resulting defensive/aversive behaviors can be evoked by odors from predators, dominant conspecifics, or the "scent" of fear from a conspecific. Fear responses can be conditioned (learned) or unconditioned (innate). Rodents will innately respond with stereotypical fear behaviors when presented with the scent of stressed or dead mice. Detection of

**FIGURE 3 | Specific innate behaviors are controlled by distinct regions of the limbic system. (A)** Sexual behaviors include activation of the vomeronasal organ (VNO), accessory olfactory bulb (AOB), and medial amygdala (MeA). Signal transduction from sensation to physical motivation is not always linear; once signal has reached the MeA, it is dispersed to a few areas: bed nucleus of stria terminalis (BNST), medial preoptic nucleus (mPN), and premammillary nucleus (PMN). The BNST will shunt signal from the MeA to the mPN. The mPN can activate appetitive behaviors (sniffing and pursuit) through innervation of the nucleus accumbens (NuAc) and ventral tegmental area (VTA). Additionally, the mPN passes information to the ventrolateral portion of the ventral medial hypothalamus (VMHvl), which in turn can initiate consummative behaviors through the periaqueductal gray (PAG) and spinal cord. Consummative behaviors such as mounting, intromission, and ejaculation can also be influenced by PMN inputs on the PAG and spinal cord. **(B)** Defensive behaviors trigger slightly different areas of the amygdala and hypothalamus depending if the stimulus is a predator or an animal of the same species (conspecific). Defense in response to a predator initiated in the AOB sends signals to the posterioventral MeA (MeApv), then to the dorsomedial portion of the ventral medial hypothalamus (VMHdm). The VMHdm will then cross-talks with the anterior hypothalamus (AH), an instance of bidirectional communication. The VMHdm, then signals to the ventrolateral portion of the dorsal PMN (PMNd), which then signals to the dorsolateral and dorsomedial PAG. Defense responses to a conspecific are initiated in the AOB which sends afferents directly to the anterior dorsal and posterior dorsal MeA (MeApd). The MeApd acts as a hub dispersing signal to three areas: mPN, VMHvl, and dorsomedial portion of the PMNd. The VMHvl will engage in cross-talk with the PMNd, which ultimately communicates with the dorsomedial and lateral PAG. **(C)** Maternal behavior circuit may begin in the AOB, which sends signal to the MeA. The MeA will send information to the mPN either directly or through the BNST. Pup avoidance is suppressed in the MeA and AH to initiate pup approach. Appetitive behaviors such as pup retrieval occur through activation of the VTA by the mPN. Consummative behaviors such as nursing are executed via activation of the PAG and spinal cord by the mPN.

these conspecific alarm pheromones evokes freezing after stimulation of the Grueneberg ganglion cells (Brechbühl et al., 2008). Rodents also have an innate fear of cat and fox odors even in lab settings without prior exposure. Additionally for mice, there is an innate fear of rats, a natural predator of mice in the wild. Exposure to rat odors may induce flight, hiding, freezing, or risk assessment behaviors in mice as part of the unconditioned fear response (Blanchard et al., 2001). These unconditioned responses suggest an evolutionary "hardwiring" of circuits for such behaviors. Upregulation of cFos expression after introduction to predator odors has been documented in the posterior ventral medial amygdala (MeApv) and VMH (Canteras et al., 1997). Similar to reproductive olfactory cues, predator odors appear to be processed more readily in the AOB as opposed to the MOB (McGregor et al., 2004). This also indicates the existence of "kairomones," a chemical emitted by one species that conveys information to another. Recent comprehensive mapping of receptor expression in conjunction with neuronal activation in the VNO has uncovered the receptor-based molecular code by which rodents identify cues associated with defense (predator and conspecific) and mating (Isogai et al., 2011). Perhaps the most striking finding of this study was the revelation that subsets of these receptors are solely dedicated to predator cues from individual species. Thus, in rodents the VNO appears to have evolved specifically to respond to cues that depend on the animal's survival in the wild, consistent with the notion that these circuits are largely hardwired. Downstream of the VNO, predator cues are processed in the AOB, and then conducted to the MeA (primarily the ventral aspect) and in turn feed directly to the dorsomedial portion of the VMH (VMHdm). However, as part of a conditioned fear response when context is dependent, cues are relayed from the hippocampus to the anterior nucleus of the hypothalamus (AH). Both the conditioned and innate fear response circuits converge on the dorsal premammillary nuclei (PMNd) of the hypothalamus which acts as an "amplifier" by triggering the pituitary and adrenal hormone release (Canteras et al., 1997; Dielenberg et al., 2001; Cezario et al., 2008) (see **Figure 3B**). Although a specific role in defense has not been found in the BNST, there are suggestions that it may modulate defensive rage and startle reflex (Dong and Swanson, 2004).

Similar endpoints, namely the PMNd, are involved in defense responses to dominant conspecifics. A common behavior test replicating a dominant conspecific scenario involves placing an "intruder" male in a "resident" male's cage. The resident male assumes a dominant role, threatening the intruder with posturing, biting, and attack. Interestingly, the medial hypothalamic circuitry of reproductive behaviors (VMH) is activated in the intruder, evidenced by increased cFos expression (Kollack-Walker and Newman, 1995; Kollack-Walker et al., 1999; Veening et al., 2005). However, in contrast to reproductive circuits, the defense response circuits converge on the "amplifier" of the predator aversion, the PMNd (Cezario et al., 2008; Motta et al., 2009). Intruders may react with passive (freezing) or active defense (rearing, boxing, or dashing away) in response to the approach of the resident. Lesioning of the PMNd results in decreases in passive defense while active defenses are maintained, suggesting the possibility that the intruder has a reduced fear of the dominant conspecific (Motta et al., 2009) (see **Figure 3B**).

## **MATERNAL CARE**

Similar to reproductive behaviors, maternal care can be parsed into appetitive and consummatory phases, with appetitive behaviors including nesting and pup retrieval while consummatory behaviors consist of pup grooming and nursing. It has been suggested that there are two mechanisms at play during maternal behaviors: activation of pup attraction and repression of pup avoidance. Pup avoidance has been observed in unprimed virgin female mice. However, the natural avoidance response in these virgins can be damaged with lesions of the MeA, thus stimulating maternal care of pups (Numan et al., 1993). Likewise, lesioning the AH results in the same behavior (Sheehan et al., 2001), suggesting that pup olfactory cues are processed in both the MeA and AH (regions associated with predator fear response) to stimulate avoidance behaviors in young virgin rodents. The opposing circuit regulating pup attraction, is seeded within the mPN of the hypothalamus. The mPN expresses receptors for estrogen, prolactin, and oxytocin, suggesting it may be a major target of hormone activity (Rosenblatt et al., 1994; Consiglio and Bridges, 2009; Ruthschilling et al., 2012). Lesions of this area decrease pup retrieval and nest building in postpartum females, and cFos has been noted to increase in this region after maternal behaviors (Numan and Smith, 1984; Champagne et al., 2003). It is very likely that activation of the mPN by hormones causes an inactivation of the anterior hypothalamic avoidance behaviors in addition to activating the VTA and NuAc to initiate the appetitive phase and the PAG-lumbosacral spinal cord to advance consummatory behaviors (see **Figure 3C**) (Lonstein and Stern, 1998).

## **CIRCUIT CONTROL AND REGULATION**

Through classical neuroanatomical approaches, we have now reached a stage at which the basic circuitry regulating reproductive, defensive and maternal care behaviors are generally established. More recent studies utilizing a combination of techniques at the vanguard of science are revealing the molecular underpinnings of circuit formation and function. For example, novel optogenetic techniques allow for the subtype-specific and temporal control of neuronal activity in order to elucidate the circuitry driving innate behaviors. In addition, we are also gaining a significantly greater understanding of not only the genes that are required for normal circuit formation and function, but also how non-cell autonomous stimuli such as hormones shape neuronal populations comprising innate circuits.

One of the first studies to correlate gene expression patterns to subsets of innate behaviors made use of reporter gene knockin methodologies to trace projections of genetically marked neuronal subpopulations (Choi et al., 2005). By gene expression analysis it was revealed that anatomically distinct subsets of MeA populations differentially express combinations of the LIM-homeodomain containing genes (*Lhx5*, *Lhx6,* and *Lhx9*), genes which are known to endow neuronal identity across the neuraxis (Shirasaki and Pfaff, 2002). Interestingly, these marked populations seperately respond to different innate behavioral cues (reproductive or defensive). Specifically, *Lhx6+* neurons in the posterior dorsal MeA (MeApd) are almost exclusively activated by reproductive olfactory cues and project to an area of the hypothalamus involved in initiating mating behaviors, the ventral lateral portion of the ventral medial hypothalamus (VMHvl). Complimentary, *Lhx6*− cells in the posterior ventral MeA (MeApv) respond to predator odors and project to an area of the hypothalamus regulating defense, the dorsal medial portion of the ventral medial hypothalamus (VMHdm). Most surprisingly, molecular mapping also revealed that predator cue-responsive *Lhx6*− cells in the MeAvl also project to areas of the hypothalamus regulating reproductive behaviors, the VMHvl, an apparent contradiction. To reconcile this discrepency a model was put forth in which predator odor-activated *Lhx6*− cells can inhibit the VMHvl, thereby suppressing reproductive behaviors, while simultaneously activating the VMHdm and initiating defensive behaviors (Choi et al., 2005).

Following this study, the same group more recently used optogenetic activation combined with pharmacological silencing of hypothalamic neurons to determine how mating and defensive behaviors are coordinated in the hypothalamus (Lin et al., 2011). Direct light stimulation of the neurons in the VMHvl expressing channel rhodopsin evokes mice to not only display the appropriate defensive behaviors to other males, but also inappropriately to females and inanimate objects (Lin et al., 2011). However, lightactivation of this circuit during consummative mating behavior will not evoke aggression. Thus, utilizing state of the art approaches; both genetic and optogenetic, these studies revealed that the VMH collectively integrates information for apparently non-compatable behaviors (e.g., mating and defense/aggression). However, at what level these context-appropriate behavioral outputs are controlled by cross-talk between VMH subdivisions remains to be elucidated. This analysis also resolves previous apparently contradictory studies, which showed that the VMH is activated by both mating and aggression (Kollack-Walker and Newman, 1995; Kollack-Walker et al., 1999; Veening et al., 2005). These tools will also most likely prove invaluable for understanding how information is gated at the synaptic level as well as which genetic networks are involved in specification and function of these subcircuits.

The neural circuitry that regulates innate behaviors, perhaps more so than other brain circuits, are dramatically shaped by endocrine factors, primarily sex hormones such as testosterone and estrogen (Simerly, 2005). Both circulating and local brain levels of testosterone and estrogen are expressed in a sex-dependent manner act to refine the neural circuits involved in sexually dimorphic behaviors (Reviewed in Hill and Boon, 2009; Wu and Shah, 2011). Major structures of the limbic circuit (e.g., amygdala, BNST, mPN) express estrogen receptors in both females and males. In females, estrogen is the primary hormone in the induction of maternal care. Specifically, virgin female rats will inhibit their aversion and stimulate attraction to pups after supplementation with estrodiol, thereby behaving more like nursing females (Fleming, 1986). The role of estrogen is especially interesting in the context of development of the male brain. Recent work has revealed that estrogen is required for the development of sexual dimorphism in the amygdala and even male-specific defensive behaviors (Wu et al., 2009). In male brains, testosterone is converted to estrogen by the enzyme aromatase, which is found in select neurons of the MeA, BNST, and hypothalamus (Ogawa et al., 1998). Circulating levels of testosterone can be controlled experimentally through gonadalectomy; however, even castrated males generate estrogen from testosterone produced in the adrenals. Therefore, only genetic deletion of aromatase in male mice eliminates estrogen action, resulting in a complete loss of aggressive behaviors against intruder males. Supplementation with estradiol in aromatase-null males soon after birth restores intermale aggression, albeit mild compared to wild type males. However, estradiol replacement one week after birth does not restore male-typical aggression in aromatasenull males (Toda et al., 2001), suggesting a developmental time window in construction of the male neural circuit. These hormones also appear to regulate neuronal plasticity in the adult (Cooke et al., 2003; Cooke, 2006; Dugger et al., 2008; Morris et al., 2008). For example, estrogen affects alterations of dendritic morphology in the MeA (Gomez and Newman, 1991), which can alter the perception of external cues (Mohedano-Moriano et al., 2007). As mentioned before, the circuit controlling reproduction and defense occupy similar limbic nuclei, and influence conflicting behaviors (sexually receptivity or aggression) to a single stimulus (male approach) in females depending on her maternal/hormonal status. Thus, an alternative hypothesis is that the hormonal state of an animal influences the connectivity, thereby affecting behaviors.

While estrogens shape the programming of sexually dimorphic circuits, testosterone acting directly via the androgen receptor is required for the activation and modulation of components of male-typical displays such as mating, territorial aggression, and urine marking. In addition to dramatic anatomical changes such as decrease in angio-genital distance and visibility of a nipple line, genetic deletion of the androgen receptor in male mice causes reduced male-typical behaviors (Juntti et al., 2010). This is in contrast to estrogen receptor-null males, which never or rarely display aggressive behaviors. Therefore, while testosterone or the androgen receptor is not necessary for establishing the circuitry required for innate behaviors, it is necessary to modulate the degree of innate sex-specific behaviors. Thus, in both sexes neuroendocrines, such as estrogen and testosterone have important, but genetically separable functions, in shaping sexually dimorphic brain circuits and related innate behavior.

While much focus has been given to the role that sex hormones play in modulating behavior and associated circuits, a number of studies have also revealed important roles for non-sex hormones. The most prominent of the neuropeptides are oxytocin and vasopressin, which are expressed within the diencephalon and function throughout numerous telencephalic structures including the amygdala and BNST (Hammock and Young, 2006). As supported by a number of experimental lines of evidence, oxytocin, and vasopressin play key roles in promoting mating and bonding (both pair and maternal) behaviors. Indeed, oxytocin administration via nasal spray is currently under clinical trials in attempt to alleviate the social withdrawal associated with autism (Guastella and Macleod, 2012). However, aside from these wellcharacterized pathways, much still remains unknown regarding how genetic pathways work in concert with hormones to regulate the full repertoire of innate behaviors. Toward bridging this gap in understanding, a recent study by Xu et al. (2012) stands out. Using unbiased microarray transcriptome screening, validated with *in situ* hybridization expression analyses, they identified a novel cohort of genes expressed in a sexually dimorphic manner in the amygdala, BNST, and hypothalamus. While many of these genes were not previously implicated in sexually dimorphic behavior, expression of many were found to be modulated directly by hormone levels.  Thus, it appears that while hormonal influences modulate sexually dimorphic gene expression, distinct genetic modules control the complete pattern of sexually dimorphic innate behaviors. The results of this study also suggest that more sensitive screening methodologies such as RNAseq, will also be fruitful in identifying other components of genetic networks regulating the full cohort of innate behaviors.

## **EMBRYONIC PATTERNING OF THE INNATE LIMBIC SYSTEM AND POTENTIAL LINK TO BEHAVIOR**

Since innate behaviors are established without prior experience, the regulatory circuitry must be established during embryonic or early post-natal stages of neurodevelopment, likely through a series of hierarchical stages of genetic programming. Below we review our current knowledge of innate limbic system development, and present a novel model in which innate behaviors are generated by a coordination of genetic expression events and environmental (hormonal) cues.

Birth dating studies in labeled neurons reveal that the vast majority of neurons that comprise the innate limbic system are generated during early embryonic neurogenesis, embryonic day 11–15 (E11–15) in mice (McConnell and Angevine, 1983). By late gestation, E18, most neurons dedicated for the limbic system (with the notable exception of subsets of olfactory bulb interneurons and hippocampal granule cells of the dentate gyrus which are generated throughout the lifetime of the animal) have migrated to their final locations in the brain and, in some cases, begun to make connections (Marín and Rubenstein, 2003; Batista-Brito and Fishell, 2009; Corbin and Butt, 2011). The early post-natal period is then primarily characterized by the elaboration of both short- and long-range connections and shaping of circuits via experience and, as described above, sex-specific hormonal levels. The embryonic events of neuronal patterning and specification of neurons throughout the entire neuraxis is accomplished via the actions of delineated sets of transcription factors, typically of the homeodomain and bHLH classes (Campbell, 2003; Wonders and Anderson, 2006; Corbin et al., 2008). These genes have been conserved through evolution and act in multiple species from fly and worm to mammals, underscoring their importance in neuronal development. As described below in more detail, embryonic developmental studies over the past decade have elucidated the "how" and "where" neurons of the limbic system are generated. Similar to what has been found in the spinal cord and forebrain, neuronal subtype identity in the limbic system appears to be established during the proliferative phase of embryogenesis before migration, suggesting this is a common mechanism used in the nervous system. This early endowment of identity implies that the remainder of development may largely be dedicated to carrying out a genetically predetermined program of migration, differentiation, synaptogenesis and maturation. Therefore, understanding development, especially the genetic mechanisms by which diverse types of neurons are specified, will likely have broad implications for understanding behaviors.

## **DEVELOPMENT OF THE MOE AND VNO**

Neurons that comprise the structures of the innate limbic system are generated within the first two weeks of gestation. The innate circuit begins with the peripheral olfactory sensory neurons, also called receptor neurons, that reside in two areas of the rostrum: MOE and VNO. In rodents, the MOE covers the surface of the convoluted ethmoid turbinates formed during the first two weeks of gestation when the nasal cavity begins to develop from the olfactory placodes, which indent forming the olfactory pits. The olfactory pits deepen and eventually fuse to form the primitive nasal cavity and ventral margins of the embryonic nasal septum between E12–13 (Herbert and Leininger, 1999). The VNO develops from bilateral invaginations of the olfactory epithelium in the ventral anterior portion of the developing nasal septum. By E15 the VNO is completely formed, however studies have suggested that it is not fully functional until after post-natal development (Coppola et al., 1993), and thus may be highly influenced by early olfactory cues.

Olfactory epithelial neurons arise from the olfactory placode and have recently been shown to be in part neural crest-derived (Katoh et al., 2011). Studies investigating lineage determination and differentiation of olfactory sensory neurons have implicated the bHLH transcription factors *Mammalian Achaete Scute Homology 1* (*Mash1*) and *Neurogenin1* (*Ngn1*) as important factors for epithelial neuronal specification. *Mash1* appears to be required for the generation of the deeper layer of the olfactory epithelial neurons, while *Ngn1* regulates genes that fine-tune the neuronal lineage to a more differentiated fate (Cau et al., 2002). *Ngn1+* progenitors will terminally differentiate into olfactory sensory neuron precursors, which then express other factors such as Neuronal cell adhesion molecules (NCAMs) that may play a role in the final stages of synapse formation (Calof et al., 2002). As a single olfactory sensory neuron matures, it will express a single olfactory receptor type, which detects a specific chemical cue (Malnic et al., 1999).  The MOE is broken into four zones (I–IV), each of which connect to respective domains in the (MOB) and have distinct transcriptional expression. For example, the transcription factor *Osp94* is expressed solely in zone 1 and 2, while *PAPS-S2* is only expressed in zones 3 and 4 of the dorsal olfactory epithelium (Tietjen et al., 2005). Similarly, the VNO is segregated into two zones or domains, apical and basal, which correlate with receptor type. Receptor neurons residing in the apical layer of the VNO express V1 receptors that primarily detect physiological state of conspecifics and predators (e.g., pregnant, stressed), while sensory neurons in the basal layer express V2R that detect sex and species signatures (male, female, fox, cat) (Dulac and Torello, 2003; Papes et al., 2010).

Axons of olfactory receptor neurons project a long distance into the brain to reach their target, the olfactory bulbs. In contrast to the peripherial olfactory epithelium, the olfactory bulbs are considered a forebrain structure and represent the most rostral aspect of the telencephalon. The olfactory bulbs arise from the rostral pallium (cortical region) of the telecephalon and can be distinguished as early as E13.5 in the mouse. The zonal specification in the MOE may act as a guide map for axons to their target glomeruli. Glomerulization or glomerulogenesis is estimated to occur over several days (E12–P7) (Royal and Key, 1999) through a hierarchical process (Miller et al., 2010), establishing a discrete topography (Luo and Flanagan, 2007). That is, olfactory sensory epithelial neurons expressing the same receptor type innervate common glomeruli in the olfactory bulb. The mechanisms by which these axons find their glomerular targets in the olfactory bulb has been suggested to be broken into two stages: general and specific targeting. General or pre-targeting from zone in MOE to appropriate domain in MOB has been shown to be influenced by zonal expression of the olfactory cell adhesion molecules (OCAMs) (Yoshihara et al., 1997; Reviewed in Yoshihara and Mori, 1997; Alenius and Bohm, 2003). The specific targeting mechanism of axons into a glomerulus has been suggested to be driven by the olfactory receptor itself where a mechanism downstream of the actual olfactory receptors enables fasciculation of axons that express similar receptors (Reviewed in Mombaerts, 2001, 2006; Imai et al., 2009; see also Yoshihara et al., 1997; Imai and Sakano, 2007, 2009). Specifically, olfactory receptors provide the means for axon-axon interaction by acting through G-coupled receptors to generate a unique level of cAMP, which subsequently regulates the expression of guidance factors: Nrp1 and Sema3A (Reviewed in Imai and Sakano, 2009; see also Imai et al., 2009). More recently the Slit and Roundabout (Robo) families of axon guidance molecules, have been demonstrated to control pathfinding and targeting of olfactory axons to glomeruli in olfactory bulb. Indeed, refined targeting of olfactory receptor axons to appropriate glomeruli is pertubed in Slit1/2- or Robo1/2-null mice (Nguyen-Ba-Charvet et al., 2008). However, how this system interacts with the finetuning of connectivity via the olfactory receptors themselves remains unclear (Cho et al., 2007; Nguyen-Ba-Charvet et al., 2008).

## **DEVELOPMENT OF THE MOB AND AOB**

The main output neurons of the MOB and AOB are the mitral cell. These neurons project to deeper brain structures through the lateral olfactory tract (LOT). Growth of axonal projections from the olfactory bulb to deeper brain regions occurs at embryonic stages: between E13 and birth. This process is concurrent with olfactory epithelial targeting of the bulb, suggesting that these guidance events are independent of each other and sensory inputs (López-Mascaraque et al., 1996). Axonal pathfinding of mitral axons to the olfactory cortex along the LOT is influenced by the function of cell adhesion molecules such as cartilage acidic protein-1B, later renamed lateral olfactory tract ushering substance (LOTUS). This occurs through the ability of LOTUS to suppress the natural repulsive activities of Nogo. LOTUS antagonistically binds Nogo receptor a1 (NRa1), thus blocking Nogo binding and allowing the LOT to fasiculate and find its target in the olfactory cortex. Deletion of LOTUS causes the defasiculation of LOT axons, an effect that is rescued by co-deletion of NRa1 (Sato et al., 2011). Many other factors including *Pax6* and ephrins also cooperate to form the olfactory circuit to the cortex (Nomura et al., 2006). In contrast, less is known about the development of olfactory projections that directly synapse in the amygdala, primarily due to insufficient markers of functionally distinct olfactory-limbic pathways. Effectors of axonal guidance from the MOB and AOB to other regions of the limbic system provide a challenging area of active research.

## **DEVELOPMENT OF THE AMYGDALA**

The development of the downstream targets of the olfactory system (amygdala and hypothalamus) has been the subject of recent intense investigation. Initial concepts into the development of these structures came primarily from comparative embryonic and post-natal anatomical studies. Although much of the amygdala and hypothalamus has been anatomically catalogued (Risold et al., 1994; Swanson and Petrovich, 1998; Swanson, 2000; Petrovich et al., 2001; LeDoux, 2007), relationships between embryonic primordia based on morphology only goes so far when attempting to correlate embryonic development to post-natal structures. This is due to the fact that many neuronal cell types within the brain are in fact generated far from the mature structures that they will eventually populate. Thus, initial hypotheses regarding simplified models of amygdala and hypothalamic development (Puelles and Rubenstein, 1993; Swanson and Petrovich, 1998) have recently been superseded by a more complex picture in which distinct embryonic progenitor zones (or niches) are dedicated for the generation of individual neuronal subtypes that subsequently migrate to these emerging structures (Marín and Rubenstein, 2003; Corbin and Butt, 2011).

With regard to the amygdala, extensive work has revealed that neuronal cell diversity is generated from two sets of progenitor pools: those that contribute neurons to multiple telencephalic structures (e.g., cerebral cortex, hippocampus) and those that are unique to the amygdala. The shared sources include aspects of the cerebral cortex, the ventrally located telencephalic ganglionic eminences [medial (MGE), lateral (LGE) and caudal (CGE)], as well as diencephalic sources (Nery et al., 2002; Remedios et al., 2007; García-Moreno et al., 2010; Bupesh et al., 2011; Cocas et al., 2011) (see **Figure 4**). Progenitor pools located within each of these domains express combinations of the homedomain and bHLH containing genes, *Lhx6*, *Nkx2.1*, *Gsx2*, *Mash1,* and *Ngn2*, just to name a few. As mentioned previously, differential expression of the LIM-homeodomain containing gene family marks anatomically segregated amygdalar efferent projections that separately regulate reproductive and defensive behaviors (Zirlinger et al., 2001; Choi et al., 2005). Interestingly, each amygdaloid nucleus expresses distinct patterns of LIM-homeodomain containing genes transiently during development. For example, the posterior dorsal medial amygdala (MeApd, associated primarily with reproductive behaviors) expresses *Lhx6* and *Lmo3*, the posterior ventral medial amygdala (MeApv, associated primarily with defensive behaviors) expresses *Lhx9*, and the dorsal anterior amygdala expresses *Lhx6*, *Lhx7,* and *Lmo3* (Remedios et al., 2004; Choi et al., 2005). Thus, the combinatorial expression patterns of LIM genes may provide a comprehensive mechanism for patterning the amygdala, reflecting a similarity with the LIM-code in the spinal cord.

In addition to these shared progenitor pools, there also exist embryonic progenitor pools that appear to be dedicated primarily for the amygdala. These include populations present at the pallialsubpallial border (PSB), the junction of apposition between the dorsal (pallial) and ventral (subpallial) telencephalon. These

**FIGURE 4 | Limbic system progenitor pools in the rodent embryonic brain.** Schematic of a coronal view of an embryonic brain at midneurogenesis (∼E14) reveals expression patterns of select transcription factors along the ventricular zones of the telencephalon and diencephalon. Combinatorial codes of transcription factor expression during embryogenesis determine the identity of neurons destined for various brain regions. A large portion of amygdala neuronal populations arise from two regions of the developing brain: the pallial-subpallial boundary (blue) and preoptic area (yellow) each of which express unique combinations of transcription factors. Areas of the hypothalamus also express regionally specific transcription factors: for example *Sim1* (orange) is expressed more dorsally to specify cells in the paraventricular nucleus (PVN), while *Nra51* (red) is expressed ventrally and specifies neurons in the ventral medial hypothalamus (VMH).

populations express combinations of the homeodomain genes *Pax6*, *Emx1*, *Gsx2,* and *Dbx1*, which collectively supply the entire population of excitatory neurons to the amygdala as well as the specialized intercalated interneuronal populations which gate fear conditioning and extinction.

Of the above-mentioned genes, the function of *Pax6* in amygdalar development has been the most explored. *Pax6* is required for *Gsx2+* cells to form correct excitatory and inhibitory neuron populations in the amygdala and olfactory bulb (populations also likely derived from the PSB) (Cocas et al., 2011). Moreover, *Pax6* cooperates with the nuclear receptor *Tailess* (*Tlx*) to form the PSB (Stenman et al., 2003). *Tlx* mutants display reductions in region-specific gene expression in the ventral-most pallial regions and corresponding malformations in lateral and basolateral amygdala. Interestingly, *Tlx* mutants also display aggressive behavior, a phenotype that is consistent with amygdala dysfunction (Monaghan et al., 1997). Moreover, haplosufficient *Pax6* mutants that express only one functional copy display autisticlike social deficits (Umeda et al., 2010), supporting an important role of these genes in amygdalar development. In addition to the PSB, the *Dbx1+* progenitor pool located in the embryonic preoptic area (POA), a ventral telencephalic domain just below the MGE, is a major source of projection neurons destined specifically for the MeA (Hirata et al., 2009). Interestingly, these neurons are homogeneous by electrophysiological and molecular criteria, and electrophysiologically and molecularly distinct from *FoxP2*+ neighboring MeA neurons (Hirata et al., 2009; Carney et al., 2010). This genetic parcellation of MeA neuronal cell types suggest that, consistent with the amygdala LIM-code (Choi et al., 2005), other genetically tagged populations may have separable functions in the processing of different innate behaviors.

## **DEVELOPMENT OF THE HYPOTHALAMUS**

A major termination area of projections from the MeA is the hypothalamus. Similar to the amygdala, the hypothalamus is a nuclear structure comprised of separate nuclei with varying connections, neuronal compositions and separable functions in processing innate information. The hypothalamus is located in and arises from the ventral diencephalon, is visible as early as E9 and is clearly distinguished from the telencephalon at E12.5 both anatomically and molecularly. Similar to other regions of the central nervous system, a number of genes encoding transcription factors and secreted protein morphogens help to pattern and determine the regional specificity of the hypothalamus (Blackshaw et al., 2010). This molecular scaffolding that delineates progenitor domains of the hypothalamus can be categorized into two alar (paraventricular and subparaventricular areas) and three basal domains (tuberal hypothalamus, premammillary area and mamillary area). These domains lie along the longitudinal axis and are influenced by secreted factors such as Shh, Wnts, BMPs, and Fgf. In response to these secreted factors, cells in different developmental zones express a temporal and spatial fingerprint of transcription factors that pattern development of the subnuclei of the hypothalamus (Shimogori et al., 2010). For example, the bHLH-containing transcription factors *Sim1* and *Neurog2* and homeodomain-containing transcription factor *Otp*, delineate the embryonic paraventriclular area, which is the primordial of the supraopto-paraventricular nuclear complex in the dorsal hypothalamus (Fan et al., 1996; Puelles and Rubenstein, 2003; Shimogori et al., 2010). Loss-of-function studies have revealed that *Sim1* is required for the correct positioning of paraventricular neurons (Caqueret et al., 2006), while *Otp*null mice fail to produce somatostatin, vasopressin, oxytocin, corticotropin-releasing hormone, thyrotropin-releasing hormone in the primordial periventricular, paraventricular, and supraoptic nuclei. These mice are not only devoid of these three hypothalamic nuclei but are non-viable after birth (Wang and Lufkin, 2000).

## **PROPOSED MODEL FOR CIRCUIT PATTERNING**

Despite the above-described circuitry and the growing understanding of developmental mechanisms governing specification and migration of neurons, the link between developmental mechanisms, circuit formation and ultimately behavior remains to be clarified. There appears to be a common strategy to generate neuronal diversity across the central nervous system, wherein the combinatorial expression of different transcription factors specifies regional- and subtype-specific neuronal identity. However, an open question is whether this process also encodes the molecular identity responsible for connectivity. Indeed, although experimental evidence is scarce, such a link has already been proposed in the spinal cord, in which a transcriptional matching code acts to instruct connections between specific sensory and motor neurons (Lin et al., 1998). Although the evidence for this within the brain is also highly circumstantial, it presents an attractive and simplified mechanism whereby developmental gene expression is utilized not only to direct cell fate, but also to pre-pattern circuit connectivity. In this model, in addition to patterning neuronal identity, key transcription factors encode subsets of genes, most likely cell adhesion molecules that would be required for limbic circuit specific connectivity (**Figure 5**). In support of this model in the innate limbic circuit are two provocative sets of observations. First, fate mapping and gene expression analyses have revealed that progenitor pools that generate neurons within known connected structures of the innate limbic system (e.g., olfactory system, amygdala, hypothalamus) express common sets of transcription factors whose general function in other parts of the brain and nervous system is to control neuronal identity. These genes include, for example, *FoxP2* and *Dbx1* (Hirata et al., 2009; Carney et al., 2010; Allen Brain atlas).

and estrogen) and neuropeptides (e.g., oxytocin and vasopressin).

Second, there are multiple classes of cell adhesion molecules that specifically mark the interconnected limbic system. These include Limbic system associated membrane protein (Lsamp) as well as sets of cadherins (Redies and Takeichi, 1993; Mann et al., 1998; Pimenta and Levitt, 2004). A cadherin matching code for limbic connectivity is especially attractive as it has recently been shown that expression of the same subclasses of cadherin cell adhesion molecules are required for establishment of axontarget matching in other systems such as retinal to midbrain projections and intra-hippocampal connections (Hirano et al., 2002; Osterhout et al., 2011; Williams et al., 2011). In conjunction, other studies have found cadherin expression patterns to be regulated by *Pax6* (Stoykova et al., 1997). Thus, perhaps cadherin (or other cell adhesion molecules) codes, initially established by restricted expression of key "selector" transcription factors in the embryonic brain, produce a layout for limbic system connectivity.

## **LIMBIC CIRCUITS AND NEURODEVELOPMENTAL DISORDERS**

In humans, the limbic system is intimately tied to emotion and social behaviors, and disruption of the genetic programming of limbic circuitry may be a prime mechanism underlying a variety of social disorders, such as autism spectrum disorders (Rodrigues et al., 2004; Amaral et al., 2008; Herry et al., 2008; Markram et al., 2008; Monk, 2008) including Fragile X and Rett syndrome (Hessl et al., 2007; Adachi et al., 2009). Therefore, using the mouse olfactory-limbic system to understand how an intricate circuit forms may greatly inform human disorders. Innate behaviors such as reproduction, aggression and fear all require assimilation of social cues to produce behaviors that ensure survival. Research in rodents and primates indicate the amygdala and surrounding anatomy play a critical role in innate behaviors and social cognition. Defects in amygdala growth, cellularity and function are consistently found in individuals on the autistic spectrum disorder (Baron-Cohen et al., 2000). Consistent with this, it will be highly informative to study the potential role of the hypothalamus in autism, a very understudied area of investigation.

In support of limbic-specific defects in autism, genes known to be involved in specific aspects of development of the limbic system have already been identified and validated as highranking autism susceptibility genes (see https://gene.sfari.org/ autdb/Welcome.do. for autism linked gene annotation). One such well-studied gene is the receptor tyrosine kinase *Met* (Campbell et al., 2006, 2007). *In vitro* studies suggest that *Met* is required for GnRH migration from the nasal placode to the hypothalamus (Giacobini et al., 2007) and *Met* expression has been detected in key limbic areas: cortex, amygdala, hypothalamus, and septum. Expression temporally peaks at P14 in rodent, a period of extensive outgrowth and synaptogenesis (Judson et al., 2009). Curiously, *Met* can decrease arbor complexity (Gutierrez et al., 2004), increase growth and excitatory synapse formation (Tyndall and Walikonis, 2006), or increase motility of interneurons (Powell et al., 2003; Martins et al., 2011) all depending on the identity of the cultured cells (cortical, hippocampal or basal forebrain). This suggests that *Met* may integrate intrinsic programs and external cues that cooperate to form functional neural networks. Moreover, massive information obtained from human genome wide association studies (GWAS) also implicates a number of cell adhesion molecules in autism, including multiple members of the cadherin family (Walsh et al., 2008). Although some of these genes may broadly regulate synapse formation and function across multiple domains of the nervous system (e.g., Neuroligin), quite interestingly others such as cadherin-10 (CDH10) appear to be limbic system specific (Bekirov et al., 2002; Wang et al., 2009). Therefore, unraveling the mechanisms of limbic system development will likely provide significant insight into the etiology of autism and related disorders of social cognition

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 01 March 2012; accepted: 04 April 2012; published online: 26 April 2012.*

*Citation: Sokolowski K and Corbin JG (2012) Wired for behaviors: from development to function of innate limbic* *system circuitry. Front. Mol. Neurosci. 5:55. doi: 10.3389/fnmol.2012.00055 Copyright © 2012 Sokolowski and Corbin. This is an open-access article distributed under the terms of the Creative Commons Attribution Non Commercial License, which permits non-commercial use, distribution, and reproduction in other forums, provided the original authors and source are credited.*

## Protocadherins, not prototypical: a complex tale of their interactions, expression, and functions

#### *Joshua A. Weiner 1 \* and James D. Jontes 2 \**

*1 Department of Biology, The University of Iowa, Iowa City, IA, USA*

*2 Department of Neuroscience, The Ohio State University, Columbus, OH, USA*

#### *Edited by:*

*Robert W. Burgess, The Jackson Laboratory, USA*

#### *Reviewed by:*

*Deanna L. Benson, Mount Sinai School of Medicine, USA George W. Huntley, Mount Sinai School of Medicine, USA Zhong-Wei Zhang, The Jackson Laboratory, USA*

#### *\*Correspondence:*

*Joshua A. Weiner, Department of Biology, The University of Iowa, 143 Biology Building, Iowa City, IA 52242, USA. e-mail<EMAIL_ADDRESS>James D. Jontes, Department of Neuroscience, The Ohio State University, 119 Rightmire Hall, Columbus, OH 43210, USA. e-mail<EMAIL_ADDRESS>
**INTRODUCTION**

The organization of functional neural circuits requires the precise and coordinated control of cell–cell interactions at nearly all stages of development, including neuronal differentiation, neuronal migration, axon outgrowth, dendrite arborization, and synapse formation and stabilization. This coordination is brought about by the concerted action of a large number of cell surface receptors, whose dynamic regulation enables neurons (and astrocytes) to adopt their proper roles within developing neural circuits. While a large number of protein families have been identified that may play roles in neural circuit formation, detailed cellular functions and, especially, molecular mechanisms have been elucidated for only a handful.

The protocadherins (Pcdhs) comprise a major family of *>*80 cadherin superfamily molecules expressed primarily in the developing vertebrate nervous system, with lower expression seen in other organs such as lung and kidney. The cadherin superfamily is a diverse collection of cell-surface molecules defined by the presence of several ∼110 amino acid extracellular cadherin (EC) motifs (Nollet et al., 2000; Hulpiau and Van Roy, 2009, 2011). The canonical members of this superfamily, the classical cadherins, are type I transmembrane proteins containing 5 EC repeats and a conserved cytoplasmic domain that interacts with the armadillo repeat proteins, β-catenin and p120ctn (Gumbiner, 2005; Takeichi, 2007; Nelson, 2008; Niessen et al., 2011). The classical cadherins mediate calcium-dependent, primarily homophilic, adhesion through interactions between their EC1 domains (N-terminal, most distal from the cell membrane).

The organization of functional neural circuits requires the precise and coordinated control of cell–cell interactions at nearly all stages of development, including neuronal differentiation, neuronal migration, axon outgrowth, dendrite arborization, and synapse formation and stabilization. This coordination is brought about by the concerted action of a large number of cell surface receptors, whose dynamic regulation enables neurons (and astrocytes) to adopt their proper roles within developing neural circuits. The protocadherins (Pcdhs) comprise a major family of cell surface receptors expressed in the developing vertebrate nervous system whose cellular and developmental roles are only beginning to be elucidated. In this review, we highlight selected recent results in several key areas of Pcdh biology and discuss their implications for our understanding of neural circuit formation and function.

**Keywords: protocadherins, cadherin superfamily, adhesion, neural circuits, Pcdh**

In a search for additional classical cadherins using degenerate PCR, Suzuki and colleagues discovered a related family of molecules, which they named "Pcdhs" (Sano et al., 1993). The Pcdhs are structurally similar to classical cadherins in that they are also type I transmembrane proteins containing 6 or 7 EC repeats, but their cytoplasmic domains are distinct and lack catenin-binding sites (Sano et al., 1993; Wu and Maniatis, 1999; Nollet et al., 2000; Vanhalst et al., 2005). Subsequently, Pcdhs have been shown to comprise a large and diverse collection of molecules, which are expressed broadly in the developing and mature vertebrate nervous system.

Pcdhs can be divided into two broad classes: the clustered Pcdhs (encoded by the *Pcdh-α*, -*β*, and -*γ* gene clusters, encompassing ∼60 genes in mammals) and the non-clustered Pcdhs (so-called δ-Pcdhs) (Hulpiau and Van Roy, 2009, 2011). In mammals, the clustered Pcdh genes lie in three tandem arrays encompassing ∼1 MB at human chromosome 5q31 and on mouse chromosome 18 (Wu and Maniatis, 1999; Sugino et al., 2000; Wu et al., 2001). Within the *Pcdh-α* and -*γ* clusters, multiple large "variable" exons encoding 6 EC domains, a transmembrane domain, and a variable cytoplasmic domain are each expressed from their own promoters and spliced to three small "constant" exons that encode a shared C-terminal domain (the *Pcdh-β* locus contains no such "constant" exons) (Tasic et al., 2002; Wang et al., 2002a; see **Figure 2B**). The largest group of non-clustered Pcdhs consists of the δ1 (7 EC domains) and δ2 (6 EC domains) sub-families, which are distantly related, yet exhibit short, conserved sequence motifs in their cytoplasmic domains (Wolverton and Lalande, 2001; Vanhalst et al., 2005).

The involvement of Pcdhs in neural circuit formation has been inferred on the basis of their structural homology to the classical cadherins, their molecular diversity and their differential and combinatorial expression by neurons and glia (Shapiro and Colman, 1999; Yagi and Takeichi, 2000; Takeichi, 2007). Genetic analysis of the *Pcdh-α* and *Pcdh-γ* clusters in mice has uncovered phenotypes that are consistent with such roles, including disrupted dendrite arborization (Garrett et al., 2012; Lefebvre et al., 2012; Suo et al., 2012), impaired synaptic development (Weiner et al., 2005; Garrett and Weiner, 2009), mistargeting of axons (Hasegawa et al., 2008, 2012; Katori et al., 2009; Prasad and Weiner, 2011), and neuronal cell death (Wang et al., 2002b; Emond and Jontes, 2008; Lefebvre et al., 2008; Prasad et al., 2008; Chen et al., 2012). Similarly, recent work has implicated several Pcdh genes in a variety of human neurodevelopmental disorders (Redies et al., 2012). However, clear cellular functions, molecular mechanisms of protein interaction, and signaling partners have yet to be determined for several Pcdh subfamilies, and definitive evidence for Pcdh roles in synaptic recognition and adhesion does not yet exist. While much remains obscure about these fascinating cell-surface molecules, their demonstrated critical importance to neural development and their potential links to human disease suggest that further elucidation of the mechanisms regulating their expression, trafficking, interaction, and signaling will generate important new neurobiological insights.

In this review, we highlight selected recent results in several key areas of Pcdh biology and discuss their implications for our understanding of neural circuit formation and function. For comprehensive reviews of the Pcdh families see the following publications Yagi and Takeichi (2000), Redies et al. (2005, 2012), Morishita and Yagi (2007), and Kim et al. (2011).

## **CLUSTERED PROTOCADHERINS: ROLES IN NEURAL CIRCUIT FORMATION**

Thus far, the best evidence in favor of a role for the clustered Pcdhs in neural circuit formation has been obtained for the *Pcdhγ* family (Wang et al., 2002b; Weiner et al., 2005; Prasad et al., 2008; Garrett and Weiner, 2009; Prasad and Weiner, 2011; Chen et al., 2012; Garrett et al., 2012; Lefebvre et al., 2012; Suo et al., 2012). *Pcdh-γ* genes are expressed widely in the CNS, and γ-Pcdh proteins are found immunohistochemically at some, though far from all, synapses (Wang et al., 2002b; Phillips et al., 2003), as well as in dendrites, axons, and perisynaptic astrocytic processes (Garrett and Weiner, 2009). Mice in which all 22 *Pcdh-γ* genes (Wang et al., 2002b), or just the 3 -most variable exons (C-type exons; Chen et al., 2012) have been deleted lack voluntary movements and reflexes, and die shortly after birth. This phenotype is likely due to severe apoptosis and neurodegeneration of spinal interneurons and concomitant loss of synapses in the late embryonic period (Wang et al., 2002b; Prasad et al., 2008; Chen et al., 2012). A reduction in synaptic density is apparently a primary function of the γ-Pcdhs in the spinal cord, and not merely secondary to the observed neurodegeneration: When apoptosis is blocked in *Pcdh-γ* null mice by the additional loss of the pro-apoptotic gene *Bax* (Deckwerth et al., 1996), neuronal survival is rescued, but both excitatory and inhibitory synaptic puncta remain reduced by 40–50% and the double-mutant mice die at birth (Weiner et al., 2005). Further, when loss of the γ-Pcdhs is restricted to astrocytes, spinal cord synaptogenesis is delayed, but with no concomitant effect on apoptosis (Garrett and Weiner, 2009). Interestingly, mice lacking only the three C-type *Pcdh-γ* exons, in a *Bax*−*/*− background, can survive past weaning, although they exhibit neurological impairments (Chen et al., 2012). This indicates that the loss of the C-type exons alone does not produce an exact phenocopy of the whole-cluster null.

In the retina, loss of all γ-Pcdhs also leads to apoptosis and synapse loss; however, in contrast to the spinal cord data, in this case blocking cell death by deletion of *Bax* does rescue synaptic density (Lefebvre et al., 2008). Conversely, when *Pcdh-γ* loss is restricted to the cerebral cortex, no excessive apoptosis is observed, but rather a major reduction in the dendritic arborization of cortical pyramidal neurons (Garrett et al., 2012). Together, these results from multiple regions of the CNS indicate that distinct neuronal types respond in different ways to the loss of the γ-Pcdhs. This is borne out by recent evidence that the γ-Pcdhs can mediate dendritic self-avoidance, in a manner very similar to that of the immunoglobulin superfamily molecule DSCAM (Fuerst et al., 2008, 2009), in both retinal starburst amacrine cells and cerebellar Purkinje neurons (Lefebvre et al., 2012). The dendritic phenotype (reduced arborization) observed in *Pcdh-γ* knockout cortical neurons (Garrett et al., 2012) and hippocampal neurons in which γ-Pcdh expression has been knocked down via RNAi (Suo et al., 2012) are not obviously consistent with such a self-avoidance role. Thus, it appears that the role of γ-Pcdhs in dendrite arborization may differ depending on the neuronal type, presumably due to a different repertoire of*cis*-interacting proteins and/or downstream signaling pathways.

The α-Pcdhs (originally termed Cadherin-related Neuronal Receptors, or CNRs) were the first of the clustered Pcdhs to be identified as synaptic molecules (Kohmura et al., 1998). The α-Pcdhs localize to developing axons (Blank et al., 2004; Morishita et al., 2004), consistent with the phenotypes subsequently observed in *Pcdh-α* mutant mice, which unlike the *Pcdh-γ* mutants are viable and fertile and do not exhibit increased neuronal apoptosis. Mice in which the *Pcdh-α* constant exons have been deleted exhibit an axonal targeting defect in the olfactory system, with axons expressing a given odorant receptor failing to coalesce on a single glomerulus in the olfactory bulb, as occurs in wildtype mice (Hasegawa et al., 2008, 2012). These disorganized axons appear to be able to form terminals and synapses at the glomeruli they contact; thus, the α-Pcdhs may be more important for axon guidance than they are for synaptogenesis (Hasegawa et al., 2008). Consistent with this, serotonergic axonal projections are also disorganized in mice lacking the α-Pcdhs, in some cases failing to penetrate the proper target area (Katori et al., 2009). Interestingly, morpholino knockdown of the *Pcdh-α* genes in zebrafish results in neuronal apoptosis, suggesting some distinct roles for the α-Pcdhs in different vertebrate systems (Emond and Jontes, 2008).

The *Pcdh-β* cluster remains the least studied of the three, perhaps because the lack of a shared constant domain among its members makes it more difficult to study. Using antibodies specific for two β-Pcdh proteins, β16 and β22, Junghans et al. (2008) found that both are present in the synaptic zones of the retina, the inner and outer plexiform layers, though only β16 was tightly localized to synapses, primarily the postsynaptic compartment. To date, no functional analysis of the *Pcdh-β* cluster has been published. However, Wu et al. (2007) have reported the generation of mice harboring large deletions within the clustered Pcdh loci, including loss of the *Pcdh-β* cluster, and presumably forthcoming analyses of such mice will yield functional data on the role of the β-Pcdhs in the nervous system.

## **CLUSTERED PROTOCADHERINS: COMBINATORIAL COMPLEXITY**

The assumption that Pcdhs are *bona fide* cell adhesion molecules, acting in a manner analogous to that of the classical cadherins, is central to most views of Pcdh function (Shapiro and Colman, 1999; Redies et al., 2005; Takeichi, 2007). Though not essential for Pcdhs to play a role in neural circuit formation, differential homophilic cell adhesion is conceptually the most straightforward hypothesis, and there is some support for this. Multiple studies have shown that γ-Pcdhs can mediate homophilic interactions in a variety of cell types; the strength of adhesion, however, is modest in comparison to that of the classical cadherins (Frank et al., 2005; Fernandez-Monreal et al., 2009; Schreiner and Weiner, 2010). However, γ-Pcdhs overexpressed in heterologous cells do not efficiently reach the cell surface unless their cytoplasmic domains are truncated (Frank et al., 2005; Fernandez-Monreal et al., 2009; Schreiner and Weiner, 2010), suggesting that in some experiments "weak" adhesion may be due to low surface delivery of the molecules, which is typically not assessed. The α-Pcdhs have not yet been found to exhibit significant adhesive activity, suggesting that they do not act as homophilic cell adhesion molecules (Morishita et al., 2006), though some may exhibit heterophilic interactions with β1 integrins (Mutoh et al., 2004). However, only one (Pcdh-α4) of the 8 α-Pcdhs that contain an integrin-binding RGD site within EC1 has been tested for binding to integrins. Furthermore, the ubiquitously-expressed Pcdh-αC1 and -αC2 do not contain such an RGD site, making it unlikely that they interact with integrins. Therefore, the role of α-Pcdhs in cell adhesion remains uncertain.

Schreiner and Weiner (2010) recently provided the strongest evidence to date that any of the clustered Pcdhs can mediate homophilic interactions, while at the same time demonstrating that the rules governing these interactions are likely to be much more complex than had been assumed. Using a quantitative, colorimetric assay for cell adhesion, these authors confirm that γ-Pcdhs mediate homophilic interactions, but go on to show that these homophilic *trans-*interactions occur between heteromeric *cis* complexes that are most likely tetramers based on their size (**Figure 1**; Schreiner and Weiner, 2010). These tetramers are formed by the 22 γ-Pcdhs promiscuously, with no apparent isoform restriction. Thus, the maximal number of *cis-*tetramers

respectively. Lines represent direct interactions reported in the literature. The dashed line indicates that a direct interaction between C-cadherin and PAPC has not yet been demonstrated. **(B)** Members of each of δ-protocadherin and γ-protocadherin sub-families have been shown to mediate trans-homophilic interactions *in vitro*. These interactions

complexity of cell interactions. In addition, mammalian α-Pcdhs contain an RGD sequence and exhibit trans-heterophilic interactions with β1-integrin. Similar RGD sequences are present in the δ2-Pcdhs, Pcdh17, and Pcdh19 suggesting that these proteins may also mediate heterophilic binding [indicated by (?)]. Heavy dashed line indicates *trans*-interactions.

that can form is 234,256 (224), though assuming a degree of functional equivalence among tetramers with the same composition (that is, assuming topological organization within the membrane is not critical) the number is more likely to be on the order of 104 (Zipursky and Sanes, 2010; Yagi, 2012).

In addition to cis-heteromers of γ-Pcdh isoforms, evidence suggests that α-Pcdh, β-Pcdh, and γ-Pcdh proteins also associate in complexes. A potential functional relationship between the α-Pcdhs and γ-Pcdhs was initially shown by Murata et al. (2004), who found that the γ-Pcdhs could facilitate trafficking of α-Pcdhs to the cell surface in transfected HEK293 cells, in which the latter rarely make it to the plasma membrane alone (Murata et al., 2004). In the absence of γ-Pcdhs, α-Pcdhs are retained in the endoplasmic reticulum and Golgi apparatus, with only low levels delivered to the cell surface (Murata et al., 2004). More recently, biochemical studies have demonstrated that α-, β-, and γ-Pcdhs can all be co-isolated by immunoprecipitation, both *in vitro* and *in vivo* (Chen et al., 2009a; Han et al., 2010; Biswas et al., 2012). The functional significance of such pan-cluster complexes remains unclear, but it is tempting to suggest that the αand β-Pcdhs could modify the homophilic specificity exhibited by γ-Pcdh multimers. If so, the resulting combinatorial explosion in adhesive interfaces would mean, in theory, that the clustered Pcdhs have the ability to endow essentially every neuron with a unique molecular identity (Yagi, 2012).

## **PROTOCADHERIN–CADHERIN INTERACTIONS**

There is increasing evidence for a relationship between the nonclustered δ-Pcdhs and classical cadherins (**Figure 1**). The Pcdh8 like molecule, PAPC (paraxial Pcdh), mediates cell sorting in Xenopus animal cap assays, which was initially taken as evidence for homophilic cell adhesion (Kim et al., 1998). More recently, Gumbiner and colleagues showed that PAPC does not itself mediate cell adhesion. Stable lines expressing PAPC failed to exhibit adhesion either in laminar flow assays or in cell aggregation assays (Chen and Gumbiner, 2006). Moreover, fusions of the PAPC ectodomain to the Fc region of IgG failed to mediate adhesion in bead aggregation assays (Chen and Gumbiner, 2006). Thus, despite the ability to effect cell sorting, PAPC does not function as a cell adhesion molecule. Chen and Gumbiner (2006) went on to resolve this apparent conflict by demonstrating that PAPC actually antagonizes adhesion by C-cadherin, although they did not demonstrate a physical interaction between the two proteins (Chen and Gumbiner, 2006). A similar antagonistic relationship was found between Pcdh8/Arcadlin and N-cadherin in cultured hippocampal neurons (Yasuda et al., 2007). Induction of Pcdh8 expression by electroconvulsive shock treatment in rats results in internalization of Ncad and removal from synaptic junctions. In this instance, there is a physical interaction of Pcdh8 with N-cadherin, as the proteins can be co-immunoprecipitated. Upon expression, Pcdh8 associates with Ncad, and *trans*-interactions mediated by Pcdh8 induce internalization through a pathway that involves the TAO2β kinase and p38 MAPK (Yasuda et al., 2007). Thus, there appears to be a close functional relationship between δ2-Pcdhs and classical cadherins.

More recently, Biswas et al. (2010) found that the δ2-Pcdh, Pcdh19, interacts with Ncad, both physically and functionally in the developing zebrafish. Knockdown of Pcdh19 in zebrafish embryos impairs neural plate convergence, resulting in malformation of the anterior neural tube. This phenotype is very similar to that of Ncad mutant embryos (Lele et al., 2002; Hong and Brewster, 2006; Biswas et al., 2010), suggesting that these molecules participate in a common pathway. Partial loss of both Pcdh19 and Ncad was shown to be synergistic, indicating that these two proteins cooperate during neural plate convergence. In addition to this functional interaction, Pcdh19 and Ncad associated physically to form a *cis*-complex (Biswas et al., 2010). In a follow up study, Emond et al. (2011) showed that secreted, epitope-tagged ectodomains of Pcdh19 and Ncad associate and can be purified from culture medium as a complex (Emond et al., 2011). When used in bead aggregation studies, this Pcdh19-Ncad complex mediates robust homophilic adhesion, although Pcdh19 on its own is not adhesive. Importantly, three lines of evidence support the idea that, within the complex, Pcdh19, rather than Ncad, is responsible for the adhesive interaction: (1) Pcdh19-Ncad complexes formed using adhesiondeficient Ncad mutants still mediate adhesion; (2) Mutations in Pcdh19 abolish adhesion by the complex; and (3) the Pcdh19- Ncad complex does not interact in *trans* with Ncad alone. Thus, Ncad appears to act as a cofactor to facilitate adhesive interactions of Pcdh19. Moreover, these results also indicate that Ncad is unavailable to mediate homophilic interactions when in complex with Pcdh19. These data suggest a model in which Ncad exists in one of two adhesive states: (1) Ncad directly mediates homophilic adhesion on its own; or (2) Ncad acts as a co-factor in *cis* to facilitate adhesion by Pcdh19 in *trans*. As another δ2-Pcdh, Pcdh17, exhibited similar behavior, Ncad may participate in multiple adhesive complexes with mutually incompatible specificities. Collectively, the data on PAPC, Pcdh8, Pcdh17, and Pcdh19 suggest that δ2-Pcdhs, at least, may affect cell adhesion (both positively and negatively) by forming complexes with classical cadherins.

There are hints that the participation of Pcdhs in homophilic interactions could be further complicated by the formation of larger macromolecular assemblies (**Figure 1**). In addition to a *cis*-interaction with δ-Pcdhs, Ncad also associates with other cell surface proteins, including Fgf receptor 2 (Williams et al., 2001), Nectin-2 (Morita et al., 2010), Cdo (Kang et al., 2003), and Robo (Rhee et al., 2002, 2007), and interacts functionally with β1-integrin (Arregui et al., 2000; Li et al., 2000). Moreover, C-cadherin, which interacts with PAPC, can also associate with the leucine-rich repeat protein, Flrt3 (Chen et al., 2009b). In addition, there is evidence that individual clustered Pcdhs or heteromeric complexes of α-, β-, and γ-Pcdhs can interact with classical cadherins (Ncad and Rcad) and δ-Pcdhs (Pcdh17) (Han et al., 2010), as well as Ret (Schalm et al., 2010) and GABA-A receptors (Li et al., 2012). Thus, the distinct cellular roles of clustered (α-Pcdh, β-Pcdh, and γ-Pcdh), non-clustered (δ-Pcdh), and classical cadherins may be difficult to define, due to the possibility of both crosstalk between and cooperation among the distinct family members. The association of Pcdhs with other gene families also suggests that a shifting protein composition within these complexes could influence the specificity of *trans*-interactions and the resulting downstream signaling pathways.

## **REGULATION OF PROTOCADHERIN TRAFFICKING**

A key element in the regulation of many cell surface proteins is the control of their trafficking, and recent data suggest this especially to be true for Pcdhs. Initial work with γ-Pcdhs found that they are present largely in intracellular organelles, with a surprisingly low proportion present on the plasma membrane (Phillips et al., 2003; Murata et al., 2004). The control of γ-Pcdh trafficking appears to be largely dependent on elements within the cytoplasmic domain: deletion of either the constant domain, or the entire cytoplasmic domain, significantly increases surface delivery of the γ-Pcdhs (Fernandez-Monreal et al., 2009; Schreiner and Weiner, 2010). In addition, the receptor tyrosine kinase, Ret, may regulate the trafficking and stability of α-Pcdh/γ-Pcdh complexes through phosphorylation (Schalm et al., 2010). Ret was shown to associate with α- and γ-Pcdhs in a neural tumor cell line and to control their protein levels: knockdown of Ret resulted in a corresponding reduction in the levels of Pcdhs (Schalm et al., 2010). Interestingly, the ectodomain of Ret contains cadherin repeats, reinforcing the notion that cadherin EC repeats may be protein–protein modules that act as scaffolds to assemble *cis*-macromolecular assemblies. These results are reminiscent of the observations of Yasuda et al. (2007), which showed that the δ2-Pcdh Pcdh8/Arcadlin associates with Ncad to induce endocytosis and removal from synaptic junctions. Thus, complex formation may be a fundamental mechanism for regulating Pcdh localization, adhesion, trafficking, and stability.

While surface delivery of the γ-Pcdhs is regulated by their cytoplasmic domains (Fernandez-Monreal et al., 2009; Schreiner and Weiner, 2010), there is also evidence that the γ-Pcdhs themselves may regulate vesicular traffic in the cell. Using correlative light and electron microscopy (CLEM), Hanson et al. (2010) showed that overexpression of γ-Pcdhs, but not Ncad, in HEK293 cells leads to the formation of elaborate membrane tubules that appear to emanate from lysosomes. Intriguingly, tubules did not form when γ-Pcdh isoforms lacking a variable cytoplasmic domain were expressed (O'Leary et al., 2011), and the width of the tubules produced was reduced when half of the ectodomain was deleted (Hanson et al., 2010). While this last result provocatively suggests that homophilic γ-Pcdh ectodomain interactions could occur within intracellular organelles, the functional significance of the tubules formed by overexpression of γ-Pcdh cDNAs in heterologous cell lines remains unclear.

## **REGULATION OF CLUSTERED PROTOCADHERIN EXPRESSION**

One of the most fascinating aspects of the clustered Pcdh families is their differential and combinatorial expression in cells of the nervous system. A decade ago, Tasic et al. (2002) and Wang et al. (2002a) concurrently identified the mechanism of *Pcdh-α* and *-γ* transcription. The sequence upstream of each variable (V) exon contains its own promoter region including a ∼20 base pair conserved sequence element (CSE) that is required for expression. Through mechanisms that are still not entirely clear, a given V exon promoter is "chosen" and transcription through the remaining portion of the *Pcdh-α* or -*γ* cluster proceeds; intervening V exons are then removed when the 5 V exon is *cis-*spliced to the three downstream constant (C) exons (Tasic et al., 2002; Wang et al., 2002a) (**Figure 2**). Although the *Pcdh-β* cluster does not contain its own C exons, each *Pcdh-β* V exon harbors a consensus 5 splice site near its end (Wu and Maniatis, 1999) suggesting the possibility of splicing to the C exons of other clusters. Such intercluster spliced transcripts, while rare, are apparently present in neurons, as are low levels of αV/γC and γV/αC hybrid transcripts produced by *trans*-splicing between separate pre-mRNA intermediates (Tasic et al., 2002; Wang et al., 2002a). Most of the *Pcdh-α, -β,* and -*γ* V exons are expressed monoallelically; though both cluster alleles are transcriptionally active in a given cell, an individual V exon promoter is only "chosen" from one of the two alleles (Esumi et al., 2005; Kaneko et al., 2006). The exception to this rule are the nearly ubiquitously-expressed *Pcdh-αC1* and *αC2* V exons, and the related *Pcdh-γ C3*, *C4*, and *C5* V exons, all of which can be biallelically expressed (Esumi et al., 2005; Kaneko et al., 2006). Single-cell RT-PCR analysis of cerebellar Purkinje cell neurons suggests that each cell expresses ∼4 *Pcdh-α* isoforms (2 of the monoallelically expressed genes plus the 2 ubiquitous 3 genes), ∼2 *Pcdh-β* isoforms and ∼7 *Pcdh-γ* isoforms (∼4 of the monoallelically expressed genes plus the 3 ubiquitous 3 genes) (Hirano et al., 2012; Yagi, 2012).

A key insight into the control of clustered *Pcdh* expression came with the discovery by Ribich et al. (2006) of two long-range regulatory elements located near the 3 end of the *Pcdh-α* cluster. These sites were identified based on sequence conservation and hypersensitivity to DNase I degradation; hypersensitive sites (HS) 5-1 lie 3 of the third α constant exon, and HS7 lies between α constant exon 2 and 3 (**Figure 2**). Addition of HS5-1 (or to a much lesser extent, HS7) downstream of a minimal promoter-*LacZ* cassette resulted in reporter expression throughout the CNS, demonstrating that this site can independently promote gene expression in regions known to express the *Pcdh-α* genes (Ribich et al., 2006). Deletion of the HS5-1 site in mice led to significantly reduced expression of most *Pcdh-α* genes: Expression of the 5 V exons, *Pcdh-α1-5*, was moderately reduced, while that of the more 3 *Pcdh-α6-12* and -*αC1* were greatly reduced (Kehayova et al., 2011; Yokota et al., 2011). The *Pcdh-αC2* gene was unaffected by HS5-1 deletion, consistent with prior *in vitro* results (Ribich et al., 2006). Deletion of the HS7 enhancer site resulted in a more moderate, but more uniform, negative effect on *Pcdhα* gene expression, with expression of all V exons significantly reduced in the cerebellum (Kehayova et al., 2011). Further HS sites downstream of the *Pcdh-γ* cluster were identified and termed HS16-20 by Yokota et al. (2011), who showed that deletion of these sites in mice led to a nearly complete loss of expression across the *Pcdh-β* cluster. Surprisingly, deletion of HS16-20 had a much less drastic effect on the more closely adjacent *Pcdh-γ* cluster; no effect was found on genes of the *Pcdh-α* cluster in these mice (Yokota et al., 2011).

The identification of these long-range regulatory sites provides a likely explanation for the results of Noguchi et al. (2009), who generated several lines of mice harboring deletions or duplications within the *Pcdh-α* cluster using targeted meiotic recombination. Across 4 lines of mice (deletion of α11-C2; deletion of α2-11; duplication of α2-10; duplication of α12-C2), the total *Pcdh-α* transcript levels, as measured by assaying the shared constant exons, remained fairly consistent (Noguchi et al., 2009).

exons are shown in black. Several DNase I hypersensitive sites (HS) that have been identified as enhancers are shown as ovals, with their reported effects on clustered Pcdh gene expression noted below. Schematic shows approximate locations along the chromosome but is not to strictly to scale. **(B)** Schematic of the mouse *Pcdh-γ* cluster showing an example pattern of gene transcription and splicing. Each variable exon has its own upstream promoter from which transcription is initiated. A long transcript through the rest of the cluster is subsequently spliced such that each transcript contains only the 5 -most variable exon

In the deletion lines, the remaining exons were upregulated, while in the duplication lines, individual exons were downregulated, to maintain expression levels. Consistently, the 3 -most V exon in each line took on the ubiquitous expression pattern normally found only for *Pcdh-αC1* and -*αC2* (Noguchi et al., 2009). This suggests that proximity to the HS5-1 and/or HS7 sites, in part, regulates expression levels of individual exons and may account for the varying expression levels across the *Pcdh-α* cluster.

Two zinc finger transcription factors recently have been identified to regulate the clustered *Pcdh* genes: neuron-restrictive silencer factor/RE-1 silencing transcription factor (NRSF/REST), and CTCF (CCCTC-binding factor). NRSF/REST binds to neuron-restrictive silencer elements (NRSEs) to repress neuronal gene expression in non-neuronal cells (Chong et al., 1995; Schoenherr and Anderson, 1995). There are multiple canonical and non-canonical NRSF-binding sites (NRSEs) within the fugu (pufferfish), mouse, and human *Pcdh* clusters (Tan et al., 2010), and deletion of NRSEs from *Pcdh* constructs causes their expression in transgenic *Xenopus* tadpoles to shift from neural-specific to ubiquitous. Kehayova et al. (2011) confirm that the HS5-1 element contains a non-canonical NRSE site, and that this site is conserved sequence element (CSE; white) and an adjacent variable exon (blue). In neurons, CTCF and Rad21 bind near the CSE and promote expression in concert with the HS5-1 enhancer element. In non-neuronal cells, NSF/REST may suppress gene expression by binding to canonical and non-canonical NRSE sites either in the promoters (*Fugu*) or within the coding sequences (mammals). Hypermethylation of clustered Pcdh promoters may also inactivate gene expression; increased methylation has been reported in various cancerous cell types and in brain following environmental stressors such as poor maternal care.

required for the suppression of mouse *Pcdh-α* promoter activity in a kidney cell line.

CTCF binding sites are found within the promoters of *Pcdh-α* V exons, as well as within HS5-1 (Kehayova et al., 2011; Golan-Mashiach et al., 2012). This is particularly interesting, as CTCF is known to mediate enhancer/promoter interactions through DNA looping (Gillen and Harris, 2011). Several experimental observations are consistent with such a role at the *Pcdh-α* locus: (1) Knockdown of CTCF with siRNAs reduces the expression of 2 assayed *Pcdh-α* genes in the HEC1-B cell line (Golan-Mashiach et al., 2012); (2) As assayed by chromatin immunoprecipitation (ChIP) from brain samples, deletion of HS5-1 leads to reduced CTCF binding at those *Pcdh-α* promoters that are strongly affected by HS5-1 deletion, but not at those that are only weakly affected (Kehayova et al., 2011); and (3) Transcriptionally active *Pcdh-α* promoters bind both CTCF and the nuclear phosphoprotein Rad21, a subunit of the cohesin complex involved in sister chromatid cohesion during mitosis. The HS5-1 enhancer also binds CTCF and Rad21, and knockdown of either CTCF or Rad21 reduces the expression of several alternatively-expressed *Pcdh-α* isoforms (Monahan et al., 2012). It will be important in the future to determine whether similar roles for NRSF/REST, CTCF, and cohesin exist at the *Pcdh-γ* cluster, and to uncover the mechanisms by which these factors collaborate to render a "promoter choice" within individual neurons.

A spate of recent papers has revealed that the clustered Pcdh genes can be epigenetically silenced by methylation, and that dysregulation of this process may underlie multiple types of cancer as well as the deleterious effects of environmental stress. Using two mouse cell lines, Kawaguchi et al. (2008) showed that methylation of each *Pcdh-α* promoter, as well as the 5 region of each V exon, correlates negatively with its expression level; experimentally inducing demethylation increased *Pcdh-α* transcription, while inducing hyper methylation decreased it. Consistent with their ubiquitous expression in neurons, the promoters of *Pcdh-αC1* and -*αC2* are hypomethylated *in vivo* (Kawaguchi et al., 2008). Studies have found that hypermethylation of CpG islands (CGIs) within *Pcdh-α* and -*β* variable exons was highly predictive of poor prognosis across a large group of neuroblastoma samples (Abe et al., 2005). However, this increased methylation does *not* result in the decreased expression of *Pcdh-β* genes in tumor samples, indicating that while *Pcdh* hypermethylation is statistically predictive of cancer outcomes, the underlying mechanism does not involve *Pcdh* expression levels *per se* (Abe et al., 2005).

A more direct link between the clustered *Pcdhs* and cancer progression has been forged by studies from Karim Malik and colleagues (Dallosso et al., 2009, 2012). Genome-wide analysis of promoter hypermethylation in Wilms' tumor (WT), a pediatric cancer of the kidney, identified the three *Pcdh* clusters in patient samples. Multiple *Pcdh-α*, *-β*, and *-γ* genes were found to be hypermethylated in WT samples; while *Pcdh-α* gene expression is not normally detectable in fetal kidney, *Pcdh-β* and *-γ* expression is, and the latter is consistently downregulated in WT, with some genes ∼90% silenced (Dallosso et al., 2009). Importantly, siRNA knockdown of *Pcdh-γ* genes in kidney cell lines leads to increased β-catenin/TCF reporter gene activity and increased expression of known target genes of the canonical Wnt pathway, which is known to be constitutively active in WT. Conversely, overexpression of *Pcdh-γ* cDNAs in WT and HEK293 cell lines leads to growth inhibition in soft agar assays (Dallosso et al., 2009). More recently, a tumor suppressor function was confirmed specifically for *Pcdh-γ C3* in colorectal adenoma and carcinoma cells: overexpression of constructs encoding γ-Pcdh-C3 suppresses Wnt and mTOR signaling and reduces colony formation in the colon carcinoma cell line HCT116 (Dallosso et al., 2012).

Together, these exciting results suggest that Pcdhs may be an important new therapeutic target in multiple types of cancer. Importantly, several of the classical cadherins have been implicated in cancer progression; for example, loss of E-cad is a common event in a variety of epithelial cancers (Cavallaro and Christofori, 2004). The demonstrated interaction between classical cadherins and members of the Pcdh family, therefore, suggests that modulation of Pcdh expression or localization could have follow-on effects on the classical cadherins that could regulate tumor progression. It will be important in future studies to reassess the vast literature on classical cadherins in cancer in light of their demonstrated regulation by Pcdhs, as well as to determine whether Pcdh expression is disrupted in various tumor cell types.

The clustered Pcdhs may also play a role in the brain's response to environmental stress, based on studies showing that poor maternal care [assessed by the frequency of maternal licking and grooming (LG) behaviors] correlates with changes in the methylation of their genes. McGowan et al. (2011) analyzed methylation, histone H3-lysine-9 (H3K9) acetylation, and gene expression patterns across 7 MB surrounding the gene encoding the glucocorticoid receptor NR3C1, a prominent target for mediating response to stress, in hippocampal samples from offspring of rat mothers who exhibited high or low LG (McGowan et al., 2011). The three Pcdh gene clusters are located within this region on rat chromosome 18, and hypermethylation of multiple Pcdh genes was observed in offspring of low LG mothers. Conversely, many of the genes across the three Pcdh clusters show significantly higher expression in offspring of high LG mothers (McGowan et al., 2011). A follow-up study showed that this pattern is conserved in humans: hippocampal samples from suicide completers with a history of severe child abuse exhibited hypermethylation across the *Pcdh* gene clusters (Suderman et al., 2012). These epigenetic studies are particularly compelling given the dependence of proper serotonergic axon targeting on the α-Pcdhs (Katori et al., 2009), and of cortical dendrite arborization on the γ-Pcdhs (Garrett et al., 2012), and indicate that the clustered Pcdhs may be critical mediators of neural circuit changes in response to environmental stress during brain development.

## **CONCLUSIONS**

The standard, *a priori* view of Pcdhs was that they simply represented an expanded complement of classical cadherin-like cell adhesion molecules. Accumulating evidence suggests that this model is incomplete. From the recent literature, several insights into Pcdhs can be gleaned. First, Pcdhs appear to function as part of larger macromolecular assemblies. The clustered Pcdhs form heteromeric *cis*-complexes that include α-, β-, and γ-Pcdhs, as well as Ret kinase, and, potentially, many other proteins (Chen et al., 2009a; Han et al., 2010; Schalm et al., 2010; Schreiner and Weiner, 2010). In addition, δ2-Pcdhs can physically associate with classical cadherins, and the Pcdh-Cad complexes appear to be important *in vivo* (Yasuda et al., 2007; Biswas et al., 2010; Emond et al., 2011). As Ret also contains cadherin repeats in its ectodomain, these results could suggest that the cadherin motif functions as a mediator of protein–protein interactions to assemble multiprotein *cis*-complexes, in addition to their known role in homophilic *trans*-interactions. Second, the role of Pcdhs in cell adhesion can be complex and indirect. Some Pcdhs may be able to act as adhesion molecules on their own, others appear to mediate adhesion only by associating with one or more cofactors or coreceptors, while still others clearly inhibit cell adhesion by antagonizing classical cadherin interactions.

When considered broadly, the recent data suggest two significant concerns: (1) Adhesion studies in reduced *in vitro* systems (including those based in heterologous cells and those utilizing purified proteins on beads or substrates) may not recapitulate the nature of adhesive interactions as they would occur in endogenously-expressing cell types *in vivo*. Thus, the individual ectodomains used in bead-based assays or the expression of molecules in heterologous cells likely will not reflect the adhesive interactions of multimolecular protein assemblies occurring *in vivo*. In the future, this should be addressed by better defining the complexes that exist *in vivo*, which would then allow for a more realistic reconstitution of complexes for *in vitro* studies. (2) Interpreting functional experiments, such as knockouts, as demonstrating a specific role for a molecule *per se* should be seen as provisional, as further work is needed to show whether the observed phenotype is due specifically and directly to the loss of the targeted molecule, or to shifts in the composition and function of multi-protein assemblies that may vary with cell-type and developmental time. It will, thus, be important to consider carefully the results of biochemical and proteomic

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experiments in reduced systems when interpreting functional studies, and to develop new strategies for dissecting this biological complexity (e.g., looking for genetic interactions in animals heterozygous for both a Pcdh and a known interactor). The recent biochemical experiments (e.g., Schreiner and Weiner, 2010; Emond et al., 2011) that have elucidated molecular mechanisms of Pcdh interactions have actually shown that it may be more difficult than initially believed to understand the cellular and developmental processes in which these molecules participate. With this in mind, we can likely look forward to many surprises as further studies of these far-from-prototypical adhesion molecules emerge.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 20 December 2012; accepted: 01 March 2013; published online: 19 March 2013.*

*Citation: Weiner JA and Jontes JD (2013) Protocadherins, not prototypical: a complex tale of their interactions, expression, and functions. Front. Mol. Neurosci. 6:4. doi: 10.3389/fnmol.2013.00004*

*Copyright © 2013 Weiner and Jontes. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

## Molecular codes for neuronal individuality and cell assembly in the brain

## *Takeshi Yagi 1,2\**

*1 KOKORO-Biology Group, Graduate School of Frontier Biosciences, Laboratories for Integrated Biology, Osaka University, Yamadaoka, Suita, Osaka, Japan 2 CREST, Osaka University, Suita, Japan*

#### *Edited by:*

*Joshua A. Weiner, The University of Iowa, USA*

#### *Reviewed by:*

*Joshua A. Weiner, The University of Iowa, USA Woj M. Wojtowicz, University of California, Berkeley, USA*

#### *\*Correspondence:*

*Takeshi Yagi, Graduate School of Frontier Biosciences, Osaka University, 1–3 Yamadaoka, Suita, Osaka 565–0871, Japan. e-mail<EMAIL_ADDRESS>
The brain contains an enormous, but finite, number of neurons. The ability of this limited number of neurons to produce nearly limitless neural information over a lifetime is typically explained by combinatorial explosion; that is, by the exponential amplification of each neuron's contribution through its incorporation into "cell assemblies" and neural networks. In development, each neuron expresses diverse cellular recognition molecules that permit the formation of the appropriate neural cell assemblies to elicit various brain functions. The mechanism for generating neuronal assemblies and networks must involve molecular codes that give neurons individuality and allow them to recognize one another and join appropriate networks. The extensive molecular diversity of cell-surface proteins on neurons is likely to contribute to their individual identities. The clustered protocadherins (Pcdh) is a large subfamily within the diverse cadherin superfamily. The clustered *Pcdh* genes are encoded in tandem by three gene clusters, and are present in all known vertebrate genomes. The set of clustered *Pcdh* genes is expressed in a random and combinatorial manner in each neuron. In addition, *cis*-tetramers composed of heteromultimeric clustered Pcdh isoforms represent selective binding units for cell-cell interactions. Here I present the mathematical probabilities for neuronal individuality based on the random and combinatorial expression of clustered Pcdh isoforms and their formation of *cis*-tetramers in each neuron. Notably, clustered *Pcdh* gene products are known to play crucial roles in correct axonal projections, synaptic formation, and neuronal survival. Their molecular and biological features induce a hypothesis that the diverse clustered Pcdh molecules provide the molecular code by which neuronal individuality and cell assembly permit the combinatorial explosion of networks that supports enormous processing capability and plasticity of the brain.

**Keywords: protocadherin, Pcdh, CNR, neuron, neural circuits, complex networks, combinatorics, complexity**

## **INTRODUCTION**

The mammalian brain is a complex multi-cellular system composed of an enormous number of cells, including neurons and glia. In the brain, the individual neurons are highly differentiated and well organized into neural networks that generate various brain functions, and the activity of each neuron reflects the encoded information.

Recent progress in neuroscience has revealed mechanisms by which many brain functions are controlled, but essential questions remain concerning the precise nature of information processing in the brain (reviewed by Buzsaki, 2010). How can nearly limitless number of information be processed by a finite number of neurons? How can such information be integrated with other information in the brain? How are different sets of information processed in parallel? The answers to these "how" questions require the existence of a basic neuronal code for information processing in the brain (reviewed by Sakurai, 1999). An individual neuron, the basic functional unit of the brain, has a specific firing activity, and is uniquely coordinated in a circuit with many other neurons in

response to specific stimuli. A single neuron can have several to 10,000 synaptic contacts on it, and therefore receive several to ten thousand inputs.

Donald Hebb hypothesized that a discrete interconnected group of active neurons, a "cell assembly," represents a distinct cognitive entity (Hebb, 1949). Although the experimental identification of these hypothesized cell assemblies proved difficult for decades, recent rapid progress in the large-scale recording from individual neurons has experimentally defined putative cell assemblies (reviewed by Buzsaki, 2010). Under Hebb's cell assembly hypothesis, a nearly limitless number of combinatorial neuronal groups can be theoretically produced from the limited number of neurons by combinatorial explosion. Thus, the "how" questions posed above can be solved, at least theoretically, by the cell assembly hypothesis. Furthermore, recent reports show that predictive neuronal activity by spontaneous firing is observed even before an event or experience happens (Kenet et al., 2003; Dragoi and Tonegawa, 2011). These findings might mean that each "cell assembly" is intrinsically predetermined before experiences are processed in the brain.

The immune system is a genetically predetermined system for recognizing external antigens (Tonegawa, 1983; Lieber, 1992). Enormous numbers of diverse immune cells are produced developmentally by the nearly random DNA rearrangement of immunoglobulin and T-cell receptor genes; these cells include the proper immune cells for responding to certain antigens (refer **Figure 7**). This system can learn and memorize a nearly limitless number of antigens, against which it produces antibodies when an animal is attacked again by the same antigen. The molecular mechanism for the predetermined immune memory system was solved decades ago, when only limited genomic information was available with random combinations. The identification of similar molecular mechanisms may explain the "how" questions of the enormous information processing capability of the brain. In particular, the molecular codes for neuronal individuality and interconnectivity are likely to be important; for example, the discovery of thousands of odorant receptors opened new avenues of investigation in the field of odorant sensory system biology (Buck and Axel, 1991).

By analyzing nerve regeneration, Langley and Sperry similarly hypothesized that there was some type of special chemical relationship between each class of nerve fiber and each class of nerve target cell (Sperry, 1963; Langley, 1895). Sperry's chemoaffinity hypothesis proposed the existence of individual identification tags that linked each axon to only specific target cells. Recent efforts to find "molecular tags" have led to the identification of "gradient molecules." Complementary gradients of Eph kinases and their ligands, ephrins, play significant roles in establishing topographically organized maps, i.e., the retinotectal map (Cheng et al., 1995; Drescher et al., 1995; McLaughlin and O'Leary, 2005). In addition, axonal guidance molecules and receptors, which guide each axon to its target cells by contactmediated and diffusible mechanisms, have been identified, and include ephrins, semaphorins, netrins, plexins, robos, slits, and others. The guidance cues act as both attractants and repellents (Dickson, 2002). In addition, specific adhesion and adhesioninducing proteins are expressed differentially in specific neuronal populations. These include the cadherins and non-clustered Pcdh (∼20 genes, Takeichi, 2007), the neurexins and neuroligins, which have a large number of alternative splicing forms (Sudhof, 2008), and the olfactory receptors (∼1000 genes, Buck and Axel, 1991), which have all been proposed as supporting evidence for (and likely contributors to) the "area code hypothesis" (Dreyer, 1998).

Recent studies reported that two large protein families, Dscam1 in insects and clustered protocadherin (Pcdh) in vertebrates, are promising candidates for the molecular code that stamps individuality and specific interconnectivity on a given neuron (reviewed by Zipursky and Sanes, 2010). In both cases, a large diversity of proteins encoded in a complex genome structure is expressed in combinatorial and random patterns by individual neurons. These proteins mediate homophilic binding and play critical roles in neural development. In particular, the clustered Pcdh family is proposed to provide the molecular basis for neuronal individuality through their combinatorial and random expression, which is conserved in vertebrates, including humans (Yagi, 2008). In this paper, I summarize recent findings about the clustered Pcdh molecules and suggest a hypothesis of candidates for the molecular code for neuronal individuality and cell assembly in the brain.

## **CLUSTERED Pcdh MOLECULES**

In 1998, the identification of a group of eight homologous transmembrane proteins, called cadherin-related neuronal receptors (CNRs) has been reported (Kohmura et al., 1998). In 1999, Wu and Maniatis found a large gene cluster in the human genome project data by performing a BLAST search for CNRs (Wu and Maniatis, 1999). A total of 52 genes, called clustered Pcdh, are encoded in the human genome at 5q31. Exons encoding extracellular, transmembrane, and short intracellular domains are arranged in three groups called Pcdh-α, Pcdh-β, and Pcdh-γ, which have 15, 15, and 22 members, respectively. The *Pcdh-α* genes include the 8 *CNR* genes discovered in mice. The Pcdh-α and Pcdh-γ genes have very large first exons that encode almost an entire molecule, and that the 3 constant exons (exons 2–4) are very small and encode only the last 125–150 amino acids, which are shared by all Pcdh-α and Pcdh-γ genes. Their large exons have multipule promoters and are *cis*-spliced to the constant exons (Wang et al., 2002a). In addition, there are alternative splicing (A and B) forms in the constant exons of *Pcdh-α* genes (Sugino et al., 2000). The Pcdh-β cluster has no constant exons. Their cytoplasmic tails are distinct sequences, but highly conserved. In mice, a total of 58 genes are arranged in Pcdh-α, -β, and -γ, which have 14, 22, and 22 members, respectively, (Wu et al., 2001).