JONATHAN C BEAN Genetic Labeling Reveals Novel Cellular Targets of Schizophrenia Susceptibility Gene ErbB4 and Neuregulin-1 – ErbB4 Signaling In Monoamine Neurons (Under the direction of LIN MEI)
Neuregulin 1 (NRG1) and its receptor ErbB4 are schizophrenia risk genes.
NRG1-ErbB4 signaling plays a critical role in neural development and regulates neurotransmission and synaptic plasticity. Nevertheless, its cellular targets remain controversial. ErbB4 was thought to be expressed in excitatory neurons although recent studies have disputed this view. Utilizing mice that express a fluorescent protein under the promoter of the ErbB4 gene, I determined in what cells ErbB4 is expressed and their identity. ErbB4 was widely expressed in the mouse brain, being highest in amygdala and cortex. Almost all ErbB4-positive cells were GABAergic in cortex, hippocampus, basal ganglia, and most of amygdala in neonatal and adult mice, suggesting GABAergic transmission as a major target of NRG1-ErbB4 signaling in these regions. Non-GABAergic, ErbB4- positive cells were present in thalamus, hypothalamus, midbrain and hindbrain.
In particular, ErbB4 was expressed in both dopamine neurons in the substantia nigra and ventral tegmental area and in serotoninergic neurons of raphe nuclei, but not in norepinephrinergic neurons of the locus coeruleus. In hypothalamus,
ErbB4 was present in neurons that express oxytocin. ErbB4 was expressed in a group of cells in the subcortical areas that are positive for S100β. These results identify novel cellular targets of NRG1-ErbB4 signaling. Finally, perfusion of
NRG1 into the medial prefrontal cortex enhanced both dopamine and serotonin release but with differing time courses.
INDEX WORDS: Neuregulin-1, ErbB4, Serotonin, Oxytocin, S100β, GABA,
Schizophrenia
GENETIC LABELING REVEALS NOVEL CELLULAR TARGETS OF
SCHIZOPHRENIA SUSCEPTIBILITY GENE ERBB4 AND NEUREGULIN-1 –
ERBB4 SIGNALING IN MONOAMINE NEURONS
by
JONATHAN C BEAN
DISSERTATION
Submitted to the Faculty of the College of Graduate Studies of
Georgia Regents University
Augusta Georgia
In Partial Fulfillment of the Requirement
For the Degree of
DOCTOR OF PHILOSOPHY
April
2015
ProQuest Number: 3729648
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TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS …………………………………………………………….vii
LIST OF FIGURES ………………………………………………………………….…ix
LIST OF TABLES …………………………………………………………………..….xii
1. INTRODUCTION ……………………………………………………………………1
1.1 Statement of a problem .……………………………………………………….1
1.2 Statement of specific aims….……………………………………………….…3
1.3 Review of literature ...…………….…………………………………………….5
1.3.1 Schizophrenia ……….…………………………………………………..5
1.3.1.1 History ……………….…………………………………………..5
1.3.1.2 Symptomology ….……………………………………………... 6
1.3.1.3 Epidemiology and Etiology ………….……………………… 14
1.3.2 Neuregulin-1 and ErbB4 …….…………………………………………27
1.3.2.1 History of Growth Factors and Their Receptors……….….. 27
1.3.2.2 Structure and Function…….………………………………… 30
1.3.2.3 Function in the Brain …………….……………………………38
1.3.2.4 Neuregulin-1 and ErbB4 Association with Schizophrenia ..44
1.3.2.5 Controversies in Expression Profile …….………………….51
2. MATERIAL AND METHODS.……………………………………………..………53
2.1 Generation of ErbB4-Reporter and ErbB4-Reporter; GAD67::GFP Mice..53
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2.2 Neuron Culture………………………………………………………………….56
2.3 Analysis of ErbB4-Positive Cells……………………………………………...59
2.4 Immunofluorescent Labeling……………………………...…………………..60
2.5 Microdialysis…………………………………………………………………….61
2.6 High Performance Liquid Chromatography (HPLC)………………………..62
2.7 Statistical Analysis……………………………………………………………..63
3. RESULTS …………………………………………………………………………...65
3.1 Generation and Validation of ErbB4-Reporter Mice………………………..65
3.2 Density of ErbB4-Positive Cells Varies Across Brain Regions……………68
3.3 Olfactory System……………………………………………………………….72
3.4 Cortex……………………………………………………………………………78
3.5 Hippocampus…………………………………………………………………...87
3.6 Basal Ganglia…………………………………………………………………...92
3.7 Amygdala………………………………………………………………………..98
3.8 Thalamus………………………………………………………………………102
3.9 Hypothalamus…………………………………………………………………108
3.10 Midbrain………………………………………………………………………114
3.11 Hindbrain and Cerebellum………………………………………………….124
3.12 White Matter Areas and Choroid Plexus……………………………….....130
3.13 Neuregulin-1 Enhances Dopamine and Serotonin Release……………134
4. DISCUSSION ……………………………………………………………………..139
4.1 ErbB4-positive Cells In the Olfactory Bulb And Deficits In Olfaction Seen In
Schizophrenia……………………………………………………………………..140
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4.2 ErbB4-positive Cells Are Enriched In the Intercalated Nucleus Of the
Amygdala and Blunted Affect Seen In Schizophrenia………………………...141
4.3 ErbB4 in GABA or Glutamatergic Neurons………………………………...144
4.4 ErbB4 In the Hypothalamus And Neuregulin-1 – ErbB4 Signaling’s
Possible Role In Metabolism……………………………………………………..147
4.5 Glia Cells Positive For ErbB4 In the Thalamus……………………………148
4.6 ErbB4 Is Selectively Expressed In Monoamine Neurons Implicated In
Psychiatric Disorders……………………………………………………………..149
4.7 Limitations of ErbB4-reporter Mice………………………………………….150
4.8 Neuregulin-1 Enhanced Monoamine Release………………………….....153
4.9 Translations Implications For Altered Neuregulin-1 – ErbB4 Signaling...156
5. SUMMARY ……………………………………………………………………… 158
6. LITERATURE CITED …………………………………………………………… 160
ACKNOWLEDGEMENTS
The path to completion of my Ph.D. has been a long tortuous journey fraught with many challenges. Along the way I have gotten support from many people whom I am immensely appreciative of.
To my Ph.D. committee: Drs. Darrell Brann, Alvin Terry, Lynnette
McCluskey, and Almira Vazdarjanova, and to my reader Dr. Albert Pan, you have been critical of my science and pushed me to do my best work and for that I am thankful. Your critiques and suggestions have improved the final product of my dissertation and of my published work.
To all the past and present members of Drs. Mei and Xiong’s labs, you have been supportive during my struggles, critical when I needed it most, and were there to celebrate with me during my successes. Special thanks to Drs.
Dongmin Yin and Chengyong Shen for their mentorship and support. To Dr.
Xiong, you have given me much needed critical feedback and many good suggestions on my project over the years.
To my mentor Dr. Lin Mei, you have always been supportive of me, never given up on me, and always pushed me to do more. I could not have hoped for a better Ph.D. mentor and I will forever be proud to say I trained with Lin Mei. I cannot thank you enough for what you have done for me and I cannot imagine being better positioned to move forward into a successful scientific career under anyone else’s direction. Thank you.
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Finally, to my mother Katherine Bean, my father Jerry Bean, and my daughter Raven Bean, pursuing a Ph.D. and a career in science has been a dream of mine but has come with many sacrifices. I realize that most of these of come at the expense of my family life and to you. For all the sacrifices you have made for me to allow me to follow this dream I will forever be grateful. For this, I dedicate the work herein to you. Thank you.
LIST OF FIGURES
Figure Page
1. Monoaminergic Systems and Their Receptors ………………………………….12
2. Cholinergic System and GABA and Glutamatergic Circuity …………………..17
3. Recombination and Meiosis ………………………….…………………………..25
4. Neuregulin-1 Isoforms, ErbBs and ErbB4 Isoforms …………………………….31
5. Neuregulin-1 – ErbB4 Signaling …………………………………………………36
6. Neuregulin-1 – ErbB4 Signaling in the Brain ………………………………..….40
7. Genotyping results for ErbB4-reporter and GAD67::GFP mice ……………….55
8. Control Data for Microdialysis and HPLC ………………………………………..64
9. tdTomato Labels ErbB4-expressing Neurons …………………………………..66
10. Unique Distribution of ErbB4-positive Cells In Brain Regions ………………69
11. ErbB4-positive Cells Are Enriched In the Glomerulus and Mitral Cell Layers of
Olfactory Bulb ……………...……………………………………….…………………74
12. ErbB4-positive Cells In Cortex Are Enriched In Layer 2/3, Are GABAergic
Interneurons and Positive For PV …………………………………………….……...80
13. ErbB4-positive Cells In Cortex and Hippocampus of Neonatal Mice Are
GABAergic Interneurons ………………………………………………………………85
14. ErbB4-positive Cells In Hippocampus Are Concentrated In Stratum
Pyramidal, Are GABAergic Interneurons and Positive For PV ………….………..88
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15. ErbB4-positive Cells In the Basal Ganglia Show Unique Distribution and Are
GABAergic Interneurons ………………………………………………………………94
16. ErbB4-positive Cells In the Amygdala Are Concentrated In the Intercalated
Nucleus ……………………………..…………………………………………………..99
17. ErbB4-positive Cells Are Enriched In Medial Habenula ……………………..103
18. S100β Was Detected In ErbB4-positive Cells In Thalamus, Hypothalamus,
Midbrain and Hindbrain …………………………………………………………...…106
19. ErbB4-positive Cells Enriched In the Paraventricular and Dorsomedial
Hypothalamic Nuclei …………………………………………………………………110
20. ErbB4-positive Cells Co-localize With Oxytocin But Not Vasopressin In the
Paraventricular Hypothalamic Nucleus ………………………………..…………..112
21. ErbB4-positive Cells In the Midbrain …………………………………………..116
22. ErbB4-positive Cells Co-localize With Tyrosine Hydroxylase In
Midbrain ……………………………………………………………………………….119
23. ErbB4-positive Cells Co-localize With Serotonin Neurons In Raphe
Nuclei ………………………………………………………………………………….122
24. ErbB4-positive Cells Do Not Co-localize With Tyrosine Hydroxylase In the
Locus Coeruleus ………………………………………………………………..……125
25. ErbB4-positive Cells In the Cerebellum and Hindbrain ……………………...127
26. ErbB4-positive Cells In the Corpus Callosum and Choroid Plexus ………...131
27. Experimental Design …………………………………………………………….135
28. NRG1 Induces Dopamine and Serotonin Release …………………………..137
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29. Intercalated Nucleus Role In Blunted Affect And Connectivity Of the
Amygdala ……………………………………………………………………………..142
30. DsRed Does Not Label Any Additional Cells …………………………………151
31. Models For Delayed Neuregulin-1 Enhanced Monoamine Release ……….155
LIST OF TABLES
Table Page
1. Antibodies Used In the Study ……………………………………………………..58
2. Density of ErbB4-positive Cells In Different Brain Regions …………………….71
3. ErbB4-positive Cells Are Enriched In Glomerular and Mitral Layers In the Main
Olfactory Bulb …………………….…………………………………………………….76
4. Even Distribution of ErbB4-postive Cells In Olfactory System …………………77
5. Slight Variation In Density of ErbB4-positive Cells In Cortex …………………..79
6. ErbB4-positive Cells In Cortex Are GABAergic Interneurons, Many of Which are PV-positive …………………………………………………………………………82
7. Cells Expressing ErbB4 at P1-7 Are GABAergic Interneurons In Cortex and
Hippocampus …………………………………………………………………………..84
8. ErbB4-positive Cell Densities Vary In Hippocampus ………………………...…90
9. ErbB4-positive Cells In Hippocampus Are GABAergic Interneurons, Many of
Which Are PV-positive ………………………………...………………………………91
10. ErbB4-positive Cell Densities Vary In Basal Ganglia…………..……………..97
11. ErbB4-positive Cells Are Enriched In the Intercalated Nucleus and Medial
Amygdala, Within the Amygdala ………………………….………………………...101
12. ErbB4-positive Cells Are Enriched In the Medial Habenula Of the
Thalamus ……………………………………………………………………………...105
13. ErbB4-positive Cells Are Enriched In PaVH …………………………………..109
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14. ErbB4-positive Cells Are Enriched In Raphe Nuclei In the Midbrain ………118
15. ErbB4-positive Cells Co-localize With Monoamines …………………..…….121
16. ErbB4-positive Cell Density Varies In Hindbrain Despite Low
Levels ………………………………………………………………………………….126
17. Slight Variation of ErbB4-positive Cell Densities In Cerebellum ………...….129
18. ErbB4-positive Cells Are Evenly Distributed In White Matter Areas ……….133
1. INTRODUCTION
1.1 Statement of a Problem
Schizophrenia is a debilitating psychiatric disorder that affects nearly 1% of the general population (National Institute of Mental Health; Gottesman and
Wolfgram, 1991; World Health Organization, 1998). In the Global Burden of
Disease Study 2010, schizophrenia was estimated to account for 12,970,000 years lived with disability and 610,000 years of life lost due to premature mortality making it one of the leading causes of disability worldwide (Whiteford et al.,
2013). The economic burden of schizophrenia in 2002 was $62.7 billion in the
United States alone (Wu et al., 2005). Given the large number of individuals affected and the substantial cost to both the individuals affected and to society as a whole, both in terms of humanistic cost and economic burden, understanding the pathophysiology at a basic level becomes a critical undertaking.
The etiology of schizophrenia is largely unknown. Many theories have been posited over the years, but none could fully account for all of the psychopathology of the disorder or account for much of the susceptibility. Studies demonstrating that schizophrenia ran in families and correlated with genetic
1
2 relatedness were pivotal in understanding the disorder (Gottesman and
Wolfgram, 1991; Cardno et al., 1999; Rijsdijk et al., 2011). As a result, several susceptibility genes have been uncovered. Two well characterized schizophrenia susceptibility genes are Neuregulin 1 (NRG1), a neurotrophic factor, and its receptor, ErbB4 (Stefansson et al., 2002; Yang, 2003; Mei and Xiong, 2008; Mei and Nave, 2014).
NRG1-ErbB4 signaling plays a critical role in neural development and regulates neurotransmission and synaptic plasticity (Huang et al., 2000; Flames et al., 2004; Woo et al., 2007; Chen et al., 2010). Despite NRG1-ErbB4 signaling’s critical roles in the brain and their involvement in schizophrenia,
ErbB4’s cellular targets remain controversial. ErbB4 was thought to be expressed in excitatory neurons (Garcia et al., 2000; Huang et al., 2000; Ma et al., 2003;
Kwon et al., 2005; Li et al., 2007; Iyengar and Mott, 2008; Barros et al., 2009;
Pitcher et al., 2011). However, this view has been disputed by others who believe
ErbB4 is predominantly expressed by gamma-aminobutryic acid (GABA)ergic inhibitory interneurons (Lai and Lemke, 1991; Vullhorst et al., 2009; Chen et al.,
2010; Fazzari et al., 2010; Wen et al., 2010; Abe et al., 2011; Neddens et al.,
2011; Ting et al., 2011). ErbB4 mRNA has also been detected in subcortical regions (Ozaki et al., 1997; Ma et al., 1999; Steiner et al., 1999; Bruce et al.,
2002; Anton et al., 2004); however, cellular targets remain unclear due to low resolution of earlier in-situ hybridization. Unfortunately, this question cannot be addressed by current antibodies against ErbB4 because of poor specificity and availability.
3
1.2 Statement of specific aims
Aim 1: Characterize the density of ErbB4-positive cells in the mouse brain.
In order to address controversies in the field and to identify novel cellular targets of ErbB4, ErbB4-reporter mice were created where a red fluorescent protein is expressed under the control of ErbB4’s endogenous promoter. ErbB4-reporter mice brains were cut into serial sections and examined for presence of tdTomato red fluorescence using confocal microscopy. Brains were divided into 11 major brain regions, which were subdivided to 173 unique regions. The number of
ErbB4-positive cells were quantified for each region and divided by that regions’ volume observed to obtain a relative density. Many results were as expected; however, several areas emerged as having potential novel cellular targets.
Aim 2: Fate map ErbB4-positive cells.
To test the idea that ErbB4-positive cells are predominately GABAergic, ErbB4- reporter mice were crossed with mice expressing a green fluorescent protein under the control of GAD67’s endogenous promoter, a marker of GABAergic neurons. Co-localization was quantified for specific areas. GABAergic interneurons could account for almost all ErbB4-positive cells in some areas, but not in some subcortical areas. In these areas, antibodies against common neural and glia markers were used to identify these cells.
4
Aim 3: Test NRG1’s ability to induce monoamine release at the synapse.
Having confirmed ErbB4’s presence in dopamine neurons and found ErbB4 in the novel cellular target of serotonin neurons, I next wanted to test NRG1-ErbB4 signaling’s function in monoamine neurons. Utilizing microdialysis in awake and free moving mice, a baseline of monoamine release was established using high performance liquid chromatography with electrochemical detection (HPLC-EC).
NRG1 was then reverse defused through the dialysis probe and monoamine release was monitored for changes from baseline levels.
5
1.3 Review of Literature
1.3.1 Schizophrenia
1.3.1.1 History
“The concept of schizophrenia is almost universally accepted, but also ill- defined” [(Andreasen, 1987), p. 10]. This is owing to the heterogeneity of the disorder and changing definitions over time. The concept of schizophrenia has been influenced by three major perspectives. First, Emil Kraepelin is often credited as the earliest to put forth a definition of schizophrenia when he differentiated dementia praecox (schizophrenia) from manic depressive madness
(bipolar disorder). Kraepelin saw the disorder as having an organic cause, a deteriorating course and causing a significant impairment to cognition
(Andreasen, 1987; Shenton et al., 2001; Tandon et al., 2013). Second, Eugen
Bleuler further refined the definition and coined the term schizophrenia, meaning split mind. Bleuler put more emphasis on the cross-sectional psychopathology rather than the course of the disease. He also recognized the heterogeneity of the disorder, proposing that it may be a group of disorders rather than a single one (Andreasen, 1987). Finally, Kurt Schneider placed increasing emphasis on a disconnection from reality (psychosis) and other positive symptoms (Tandon et al., 2013). The three perspectives have influenced both the American Psychiatric
Association’s Diagnostic and Statistical Manual of Mental Disorders’ (DSM) and the World Health Organization’s International Classification of Diseases’ (ICD) definitions of schizophrenia.
6
Historically, the DSM and ICD have not always been in agreement, taking more or less influence from the aforementioned perspectives. However, since the release of DSM-III in 1980 (American Psychiatric Association, 1980), the two manuals have largely agreed on the definition of schizophrenia (Andreasen,
1987; Tandon et al., 2013). The current definitions have a high level of diagnostic stability with 80-90% of those who receive a diagnosis of schizophrenia retaining that diagnosis up to 10 years later (Haahr et al., 2008; Bromet et al., 2011).
Schizophrenia is recognized to be heterogeneous with no one affected individual presenting with all of the symptoms. Classically, DSM and ICD tried to capture this heterogeneity by classifying schizophrenia into several subtypes. However, it was found the subtypes did not run in families nor were they stable over time.
One patient may be classified in one subtype during the initial diagnosis and be classified in another at a later time. For these reasons, DSM-5 and proposed changes to ICD-11, to be published in 2017, have eliminated these subtypes
(American Psychiatric Association, 2013; Tandon et al., 2013).
1.3.1.2 Symptomology
Schizophrenia is characterized by three broad classes of symptoms: positive symptoms, symptoms present in affected individuals that are not present in unaffected individuals, negative symptoms, those that missing in affected individuals that are present in unaffected individuals, and cognitive impairments.
The positive symptoms include delusions, false beliefs that are held even in the presence of overwhelming evidence of their falsehood, hallucinations, perception
7 of a sensation without a source, and disorganized speech (World Health
Organization, 1992, 1998; American Psychiatric Association, 2013).
Common delusions include those of persecution, where an affected individual may believe that others are plotting against them, those of grandeur, where one may believe they are much more important or powerful than they really are, and delusions of reference, where one finds personal significance in innocuous events or coincidences (World Health Organization, 1992, 1998).
Delusions of persecution can involve the schizophrenic believing that government agencies like the federal bureau of investigation (FBI) are tracking their activities. They may believe that co-workers or classmates are deceiving, mocking, or generally treating them unfairly. The schizophrenic experiencing this type of delusion can become paranoid and may go to great lengths to protect themselves from their perceived persecutors. Grandiose delusions have common themes of fame or of religion. The affected individual may believe that they possess exceptional talent or even supernatural powers. Some patients may believe that they are God or other type of deity. Schizophrenics suffering from delusions of reference find personal meaning in everyday events. The patient may believe a news anchor is talking directly to them and that the things being said on television or radio are meant specifically for them. Alternatively, they may interpret the actions of strangers in public, such as a head nod, as a secret code meant for them. Another common delusion is one of thought insertion, control, or broadcasting. Here the patient believes that others are controlling their thoughts
8 by removing or inserting thoughts or that their thoughts are being broadcast to others against their will (Lane, 2015b).
Hallucinations in schizophrenics are usually auditory in nature, but can involve other sensory modalities. The auditory hallucinations can present as a running commentary on one’s daily activities that would usually be critical in nature or as two or more voices conversing with each other (World Health
Organization, 1992, 1998). These can be incredibly disturbing to both the patient and those around them. The affected individual may have a hard time concentrating and thinking clearly. The voices may implore the patient towards not taking their medication or towards suicide or other types of violence (Lane,
2015a). Imaging studies suggest auditory hallucinations may occur from not only an over activated auditory cortex in the temporal lobe, but also over activation in the thalamus and hippocampus (Silbersweig et al., 1995; Shergill et al., 2000).
Although less common than auditory, visual hallucinations are the second most common type reported by schizophrenics. Patients report everything from bugs crawling on walls to imaginary people following them around. Less common are hallucinations of olfaction, gustation, or somatosensation (Lane, 2015a).
Negative symptoms can include poverty of speech (alogia), poverty of content while speaking, diminished emotional expression (blunted affect), reductions in sociality, a loss in ability to experience pleasure (anhedonia), and loss of drive or motivation (avolition) (World Health Organization, 1992, 1998;
American Psychiatric Association, 2013). A patient presenting with alogia will make very little to no spontaneous conversation and when asked questions will
9 give one or two word answers. This requires the party trying to converse with the patient to ask many follow up questions to acquire the desired information. What might be answered in one or two questions in normal conversation may take five or more with this patient. Poverty of content in speech is quite the opposite where the patient may ramble on for an extended period of time without making any meaningful points. The schizophrenic with blunted affect may report normal experience of emotion, but be unable to express that emotion outwardly.
However, studies have demonstrated that schizophrenics suffering with blunted affect often are impaired in judging the emotional state of others, suggesting a deeper impairment (Gur et al., 2006). Anhedonia is often related to the schizophrenic’s lack of ability to gain pleasure from social experiences, as well as every day experiences, and may be related blunted affect. Indeed the two appear to have a neurological basis, with schizophrenics showing less activation of areas of the amygdala and striatum, brain areas related to emotion and reward, while being shown pictures of emotional words and faces (Dowd and Barch,
2010). Finally, these impairments may contribute to avolition in the patient.
Schizophrenic patients often lack the drive to complete everyday task and show less activity in brain areas associated with reward and drive during an incentive delay task (Mucci et al., 2015).
Schizophrenics are affected in attention, learning and memory tasks, abstract thinking, and cognitive flexibility. While the cognitive impairments are not part of the diagnostic criteria, they are considered one of the most debilitating aspects of the disorder, as they are treatment resistant and persistent (Spohn
10 and Strauss, 1989). Furthermore, greater than 70% of schizophrenic patients suffer from reduced cognitive abilities (Palmer et al., 1997). These impairments appear to be present during the prodromal phase of the disorder and are even predictive of who is susceptible to later developing schizophrenia (Jones et al.,
1994; David et al., 1997). The neurological basis of deficits in executive function may stem from one of the better characterized neurological abnormalities, hypofrontality (Hazlett et al., 2000; Pachou et al., 2008; Minzenberg et al., 2009;
Liemburg et al., 2015). This is a phenomenon where the frontal cortex, more specifically the prefrontal cortex, is less active during tasks requiring executive function.
Schizophrenia is a life-long disorder that can be characterized into prodromal, active, and remission phases. The prodromal phase describes the portion of the patient’s life before they receive the diagnosis of schizophrenia.
Retrospective analyses often reveal that the patients were suffering from negative or cognitive symptoms during this period (Jones et al., 1994; David et al., 1997). The active phase describes a period where the patient is presenting with symptoms that warrant the diagnosis of schizophrenia. Positive symptoms of delusions and or hallucinations are most likely to alert others of the patients’ need for psychiatric care (World Health Organization, 1992, 1998; American
Psychiatric Association, 2013). These symptoms are readily controlled with either first generation antipsychotics that work primarily by blocking dopamine type 2
(D2) receptors or second generation antipsychotics that work by antagonizing both D2 and serotonin (5-HT) 2A receptors. Both receptors are G-protein coupled
11 receptors (GPCR) with D2 being a Gαi, primarily inhibiting cAMP signaling and 5-
HT2A being a Gαq, primarily increasing the release of intracellular calcium stores via IP3 signaling (Figure 1A and B). However, it is debated whether the actions on 5-HT2A receptors are important for second generation antipsychotics’ effects
(Kapur and Seeman, 2001; Seeman, 2002). After successfully treating positive symptoms patients are considered to be in remission. This may be better termed as residual phase as many negative symptoms and cognitive deficits may persist. Additionally, while second generation antipsychotics were developed to alleviate side effects of first generations’, they still have many of their own. These include: weight gain, diabetes, metabolic syndrome, sexual dysfunction, osteoporosis and tardive dyskinesia (Millier et al., 2014). These can often lead to noncompliance with taking medication and in turn relapse. The disorder then becomes a life-long cycle of remission and relapse.
12
Figure 1. Monoaminergic Systems and Their Receptors.
(A) Dopaminergic system. Dopamine neurons are located in the substantia nigra par compacta (SNc) and the ventral tegmental area (VTA) in the midbrain. They project to the caudate and putamen (CPu), hippocampus, and frontal cortex.
Dopamine acts on two major types of receptors D1 type (D1R, D5R), that are
GPCRs, coupled to Gαs subunit, act primarily by stimulating cAMP signaling cascades, and are excitatory, and D2 type (D2R, D3R, and D4R) that are coupled to Gαi, act primarily by inhibiting cAMP signaling, and are inhibitory.
Typical and atypical antipsychotics, like haloperidol and clozapine respectively, can antagonize D2R and other D2 type receptors. (B) Serotonergic system.
Serotonin neurons are located in the dorsal raphe (DR) and other raphe nuclei in the mid and hindbrain. They heavily innervate the hippocampus, thalamus, hypothalamus, cerebellum, and the cortex. Serotonin receptors 5-HT4, 6, and 7R are GPCRs coupled to Gαs, 5-HT1 and 5R are coupled to Gαi. 5-HT2R is coupled to Gαq, acts primarily by increasing the release of intracellular calcium
(Ca 2+ ) stores via IP3 signaling, and are excitatory. 5-HT3R is a ligand gated ion channel that is permeable to the influx of sodium (Na +) and Ca 2+ and are excitatory. Atypical antipsychotics can antagonize 5-HT2. ( C) Norepinephrinergic system. Norepinephrine neurons are located in the locus coeruleus (LC) in the hindbrain and heavily innervate thalamus, hypothalamus, cerebellum, and cortex.
Norepinephrine receptors are GPCRs, α1R coupled to Gαq, α2R coupled to Gαi and β1, 2, and 3R coupled to Gαs. Brain images were a gift for participation in an imaging study at the University of South Carolina.
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1.3.1.3 Epidemiology and Etiology
There are many theories surrounding the epidemiology and etiology of schizophrenia. Disruptions in various neurotransmitters, environmental insults, and genetic susceptibilities are among the better evidenced. One of the first theories of disruption in neurotransmitters came through serendipity. The drug chlorpromazine, a potent antihistamine, was being used as a postoperative analgesic, but was found to have a calming effect on patients. Soon it was being used on psychiatric patients to lessen combativeness. In addition to the sedating effect, psychotic patients were reporting a subsiding of auditory hallucinations
(Delay et al., 1956; Seeman, 2007). Haloperidol was introduced soon after this, having similar but more potent actions than chlorpromazine (Granger and Albu,
2005). However, the new class of antipsychotic drugs was causing profound motor side effects that resembled Parkinson’s disease.
While some of the earlier theories posited that first generation antipsychotics may produce their effects through blocking monoamine receptors, norepinephrine, dopamine, and serotonin (Figure 1), (Carlsson and Lindqvist,
1963, 1967), attention began to shift more prominently toward dopamine after being proposed by van Rossum in 1966 (van Rossum, 1966; Baumeister and
Francis, 2002; Seeman, 2007). Research conducted in the mid-1970s demonstrated that therapeutic doses of antipsychotics inversely correlated with these drugs’ abilities to bind and block dopamine receptors (Seeman et al., 1975;
Seeman and Lee, 1975; Creese et al., 1976a, b; Seeman et al., 1976b; Seeman et al., 1976a). Additional evidence supporting the dopamine theory of
15 schizophrenia includes psychoactive drugs that work through the dopaminergic system. Amphetamine (blocks and reverses dopamine reuptake transporter) can produce behavioral phenotypes in animal models and in people that resemble some of the symptoms of schizophrenia (Angrist and Gershon, 1970; Bell, 1973;
Segal and Mandell, 1974). These drugs can also exacerbate clinical symptoms in schizophrenics (Wolkin et al., 1994). Postmortem studies revealed that while unaffected individuals had a normal or classic bell curve distribution of D2 receptor densities, schizophrenics had a bimodal distribution. Half of the patients had approximately the same densities as controls while the other half had roughly twice the density in striatum (Seeman, 1987). Positron emission tomography (PET) done in live patients suggest that D2 receptors have a greater occupancy in striatum and may be correlated to decreased activity in frontal cortex (Abi-Dargham et al., 2000; Meyer-Lindenberg et al., 2002). The major shortcoming of the theory is that it can only account for the positive symptoms of the disorder. The neuroleptics affecting D2 receptors do little to treat negative or cognitive symptoms of schizophrenia (Spohn and Strauss, 1989).
Modest support for a serotoninergic theory of schizophrenia comes from
5-HT2A receptor agonists’, like lysergic acid diethylamide (LSD) and psilocybin, the psychoactive component of hallucinogenic mushrooms, ability to model several aspects of the disorder. These compounds are able to elicit agitation, anxiety, and sensory gating impairment in both animal models and humans, as well as, induce hallucinations in humans (Hyde et al., 1978; Braff and Geyer,
1980; Vardy and Kay, 1983; Schmid et al., 2014). However, hallucinations
16 caused by 5-HT2A agonists are visual in nature, while those occurring in schizophrenia are more often auditory than they are visual (World Health
Organization, 1992, 1998; American Psychiatric Association, 2013). Although it is debated whether 5-HT2A receptors mediate the effects of second generation antipsychotics like clozapine, risperidone, and olanzapine, they have a higher affinity for 5-HT2A receptors (figure 1B) than they do for D2 receptors (Altar et al., 1986; Stockmeier et al., 1993; Seeman, 2002). However, studies utilizing
PET and postmortem studies have shown deficits in 5-HT2A receptor density in schizophrenic patients (Laruelle et al., 1993; Ngan et al., 2000). This leaves open the possibility that at least part of their therapeutic effects are conferred through
5-HT receptors. The notion is supported by 5-HT2 antagonists used in preclinical trials as an add-on treatment, improved either negative symptoms or cognitive deficits (Chaudhry et al., 2002; Poyurovsky et al., 2003; Akhondzadeh et al.,
2008).
Schizophrenics have an exceptionally high incidence of smoking tobacco, with estimates between 65-85% (Hughes et al., 1986; de Leon et al., 1995;
Vanable et al., 2003). Some have postulated that schizophrenics use nicotine
(nicotinic acetylcholine receptor agonist) as a way to self-medicate possibly to combat cognitive deficits of the disorder. Nicotine use has been shown to normalize some deficits like sensory gating deficits (Adler et al., 1993; Leonard et al., 1998). A number of abnormalities have been observed in the cholinergic systems of schizophrenics (Figure 2A).
17
(Mei and Nave, 2014)
Figure 2. Cholinergic System and GABA and Glutamatergic Circuity.
(A) Cholinergic system. Cholinergic neurons are present in the caudate (C), putamen (Pu), and nucleus basalis (NB) in the forebrain. They project throughout the cortex. Cholinergic neurons are also present in the reticular formation in the hindbrain projecting to the thalamus. Acetylcholine (ACh) acts on both ionotropic nicotinic ACh receptors (nAChR) as well as GPCR muscineric ACh receptors
(mAChR). NAChRs are permeable to Na + and Ca 2+ ions and are excitatory. M1 mAChR can couple to Gαs, i, or q. M2 and 4 couple to Gαi whereas M3 and 5 to
Gαq. Nicotine can serve as an agonist for nAChR. ( B) GABA and glutamatergic circuitry. Glutamatergic neuron in orange, PV-positive GABAergic basket cell in purple, PV-positive GABAergic chandelier cell in blue, and GABAergic Marinetti interneuron in green (Mei and Nave, 2014). Glutamatergic neurons form both local and projecting circuits, whereas GABAergic interneurons are local providing inhibition. GABA acts on GABA-A receptors that are ionotropic, permeable to chloride (Cl -) influx, and on GABA-B receptors that are GPCR. GABA-BR often exists as dimers and is coupled with Cl - channels to promote inhibition.
Glutamate acts primarily on AMPA, kainate, and NMDA ionotropic receptors permeable to Na + and Ca 2+ ions. NMDA receptors are blocked by magnesium ion
(Mg 2+ ). After the cell is partially depolarized through AMPA or kainate receptors the Mg 2+ is removed and NMDA can be activated. Glutamate can act on mGluR
GPCRs that often exist as dimers and can be coupled to ionotropic glutamate receptors. MGluR1 and 5 can promote excitation whereas mGluR 2-4 and 5-8 can inhibit it. Ketamine can act as an NMDA agonist.
19
Postmortem studies have found altered levels of choline acetyltransferase
(ChAT) (an important enzyme for the synthesis of the neurotransmitter acetylcholine), in patients’ brains (Bird et al., 1977; McGeer and McGeer, 1977;
Karson et al., 1993). Magnetic resonance spectroscopy has demonstrated increased levels of acetylcholine in several areas of schizophrenics’ brains
(Bustillo et al., 2002; Theberge et al., 2004). The increased levels of acetylcholine observed may be a compensatory mechanism to deal with cholinergic receptor deficiencies. The α7 subunit of nicotinic receptors has garnered special attention. A number of studies evidence decreased expression in postmortem brains from schizophrenics (Freedman et al., 1995; Leonard et al.,
1998; Court et al., 1999; Guan et al., 1999). However, both first and second generation antipsychotics are known to decrease nicotinic receptors (Terry et al.,
2003; Terry et al., 2005; Terry et al., 2006) making the aforementioned studies difficult to interpret.
Disruptions in GABAergic (main inhibitory neurotransmitter) circuitry
(figure 2B) has also been proposed to account for the pathophysiology of schizophrenia and the theory has gained popularity more recently. Reduction in the 67 kDa isoform of Glutamic acid decarboxylase (GAD67), an enzyme responsible for GABA syntheses, is one of the more reproducible findings.
Reductions have been noted in prefrontal cortex (PFC) (Bird et al., 1977;
Akbarian et al., 1995; Guidotti et al., 2000; Hashimoto et al., 2008b), cingulate cortex (Woo et al., 2004), motor and visual cortices (Hashimoto et al., 2008a), and hippocampus (Benes et al., 2007). Some studies have implicated the
20 reduction in GAD67 specifically to parvalbumin (PV) expressing, fast-spiking interneurons (Hashimoto et al., 2003; Curley et al., 2011). Reductions in PV itself have also been noted (Woo et al., 1997; Hashimoto et al., 2003), as well as, possible reduction in PV-positive interneurons (Beasley et al., 2002; Reynolds et al., 2002; Konradi et al., 2011). PV interneurons are uniquely positioned to influence primary glutamatergic neurons, with PV basket interneurons synapsing perisomatically and PV chandelier interneurons synapsing at the axon initial segment (figure 2B). It has been demonstrated that PV interneurons play an integral role in regulating gamma oscillations (Buzsaki and Draguhn, 2004;
Cardin et al., 2009; Sohal et al., 2009). Gamma oscillations are disrupted in schizophrenic patients (Kwon et al., 1999; Vierling-Claassen et al., 2008; Wilson et al., 2008) and this is thought to underlie many of their cognitive deficits
(Gonzalez-Burgos et al., 2011).
Glutamatergic (main excitatory neurotransmitter) N-methyl-D-aspartate
(NMDA) receptor antagonists, phencyclidine (PCP), and ketamine have been used to model schizophrenia (figure 2B). They better approximate schizophrenia’s symptomology than dopamine agonists producing not only hallucinations, but also illogical thought processes, poverty of speech, flattened affect, and cognitive deficits (Rosenbaum et al., 1959; Cohen et al., 1962; Krystal et al., 1994; Malhotra et al., 1996; Malhotra et al., 1997). This led to a hypo- glutamatergic theory of schizophrenia. The theory is supported by aforementioned phenomenon of hypofrontality (Hazlett et al., 2000; Pachou et al., 2008; Minzenberg et al., 2009; Liemburg et al., 2015) and disruptions seen in
21 gamma oscillations (Kwon et al., 1999; Vierling-Claassen et al., 2008; Wilson et al., 2008). However, several studies have demonstrated increased cerebral blood flow to frontal and or cingulate cortices following ketamine administration (Lahti et al., 1995; Breier et al., 1997; Holcomb et al., 2005). These results can partially be explained by findings that NMDA antagonists preferentially antagonize NMDA receptors on GABAergic neurons at non-sedating doses (Li et al., 2002;
Homayoun and Moghaddam, 2007). This then leads to disinhibition of principal glutamatergic neurons and thus causes increased cerebral blood flow.
Accordingly, NMDA antagonists’ line of evidence may be more supportive of
GABAergic dysfunction theory of schizophrenia rather than the hypo- glutamatergic theory.
A number of brain abnormalities have been noted among schizophrenics.
However, not all schizophrenics have these abnormalities and researchers cannot agree on their locations. A meta-analysis of 15 published volumetric imaging studies found that among 50 studied brain regions, nearly all had been implicated by at least one study (Honea et al., 2005). Only left superior temporal gyrus and the left medial temporal lobe appeared to be reduced in more than
50% of the studies examined. Of note, even the best evidenced regions did not show differences in 30-40% of the studies (Honea et al., 2005). One of the better evidenced abnormalities is an increase in lateral ventricles size in schizophrenics
(Andreasen et al., 1990; Andreasen et al., 1994; Cannon et al., 1998; Juuhl-
Langseth et al., 2012; van Erp et al., 2014). However, even this is debated as not all studies have replicated it (Shenton et al., 1992; Cahn et al., 2002b). Some
22 have argued that differences in cortical and lateral ventricle volumes correlate to length of illness and length of antipsychotic treatment, adding a layer of complexity that makes interpretation of differences difficult (Cahn et al., 2002a; van Haren et al., 2008; Fusar-Poli et al., 2013).
A neurodevelopmental theory of schizophrenia posits that prenatal and perinatal environmental insults can lead to or make one more susceptible to developing schizophrenia. It may do so by either causing subtle brain abnormalities or disruptions in neurotransmitters and their receptors sometimes seen in schizophrenia. Significant associations between obstetric complications and schizophrenia have been made in the past with overrepresentations in pregnancy, fetal development, and birthing complications (Verdoux et al., 1997;
Jones et al., 1998; Cannon et al., 2002b). However, even when multiple complications are pooled, they can only account for a slight increase in prevalence (Cannon et al., 2002a). Winter births are also overrepresented in schizophrenics by 5-8% (Torrey et al., 1997; Martinez-Ortega et al., 2011). That is to say if one expected 25% of the population to be born in the quarter of the year that is winter, up to 33% of schizophrenics are born during this season. The excess birth rate for schizophrenics seen in winter calculates to only a minor increase in susceptibility to the disorder, 1.3%, assuming 1% for the general population.
A proposed mechanism is that mothers of winter-born children may be exposed to influenza or other viruses during their pregnancy. Increased rates of schizophrenia have been seen with maternal exposure to influenza, evaluated as
23 major influenza outbreak at the time and location (Adams et al., 1993; Takei et al., 1995; Takei et al., 1996) or increased antibody titers against influenza and other common viruses seen in mothers whose children later go on to develop schizophrenia (Buka et al., 2001; Brown et al., 2004). It has even been suggested that Toxoplasma gondii , a common parasite carried by cats, can increase susceptibility to schizophrenia, as antibodies against the parasite have been found in a higher percentage of schizophrenics than the general population
(Yolken et al., 2001; Leweke et al., 2004), and one study finding schizophrenics were more likely to have had a pet cat as a child (Fuller Torrey et al., 2000). Viral and parasitic infections may account for a small increase in susceptibility, but are controversial as several studies have failed to find the connection between influenza and schizophrenia (Torrey et al., 1988; Cannon et al., 1996; Morgan et al., 1997) or between Toxoplasma gondii and schizophrenia (Buka et al., 2001;
Xiao et al., 2010; Khademvatan et al., 2014).
A major breakthrough in understanding the etiology of schizophrenia came when it was noted that not only did schizophrenia run in families, but the susceptibility was associated with relatedness and not being raised by a schizophrenic. It was noted that having a third degree relative, 12.5% shared genes, with the disorder double one’s odds of also having schizophrenia, while having a second degree relative, 25% shared genes, puts one at 2-6x the risk.
Having a first degree relative with schizophrenia, 50% shared genes, puts one at
6-17x risk, and finally, when one identical twin has the disorder, there is a 48% chance the other will also have it, compared to 1% for the general population
24
(Gottesman and Wolfgram, 1991). Meta-analysis from 12 separate studies comparing mono and dizygotic twins concluded with 95% confidence that schizophrenia is 81 ± 8.5% heritable, while environmental effects shared by a twin pair accounted for 11 ± 8% of susceptibility. The same meta-analysis report looking at 5 adoption studies concluded that if an adoptee had schizophrenia, it was 5x more likely that their biological parents also had schizophrenia than their adoptive parents (Sullivan, 2005).
With a clear heritability component accounting for a large portion of susceptibility to schizophrenia, researchers began looking for chromosomal loci and genes that may confer risk to the disorder. Early reports focused on genetic linkage, a technique that requires building large and complex pedigrees for families with a history of schizophrenia and looking for chromosomal loci passed on to affected individuals more often than expected. To understand the concept, one must remember that through recombination and meiosis we inherit a mosaic copy of parental chromosomes. Genes or loci that are close together are less likely to be separated through this process, and the probability of a crossover event can be calculated using the distance between them (Figure 3).
During the 1990s, several chromosomal loci were identified using this method, including loci on 2q (Levinson et al., 1998; Paunio et al., 2001), 6p
(Antonarakis et al., 1995; Straub et al., 1995; Group, 1996), 8p (Pulver et al.,
1995; Group, 1996; Kendler et al., 1996; Blouin et al., 1998), 10q (Faraone et al.,
1998; Schwab et al., 1998), 13q (Lin et al., 1995; Blouin et al., 1998; Shaw et al.,
1998), and 22q (Coon et al., 1994; Pulver et al., 1994; Gill et al., 1996).
25
Figure 3. Recombination and Meiosis.
Diagram depicting somatic cells as diploids having two copies of each chromosome, one from the father in blue and one from the mother in pink. During mitosis chromosomes are duplicated, then divided into two new daughter cells.
During the process of meiosis, chromosomes are first duplicated. After this chromosomes may cross over and recombine exchanging portions. The cell then divides twice with each of four daughter cells getting one copy of each chromosome (haploid) that may be a mosaic of the two original chromosomes.
26
Subsequent susceptibility genes were identified by fine mapping, and associations with polymorphisms and schizophrenia were noted in the general public. Some susceptibility genes support earlier theories like disruption in monoaminergic neurotransmitter systems, including D2R (Arinami et al., 1996;
Glatt and Jonsson, 2006), 5-HT2A (Williams et al., 1996; Vaquero Lorenzo et al.,
2006), and catechol-O-methyl transferase (COMT) (Zinkstok et al., 2008; Voisey et al., 2012) or in the GABAergic system with polymorphism identified in GAD67
(Addington et al., 2005; Straub et al., 2007). Others susceptibility genes may be involved in synaptic plasticity like dysbindin (Straub et al., 2002; van den Oord et al., 2003; Li et al., 2005).
Schizophrenia has thus emerged as a multi-causal, with genetics accounting for much but not all of the susceptibility, and polygenic disorder, with no one gene found to be responsible. With genetics accounting for much of the susceptibility to schizophrenia, understanding the genes that contribute to the disorder, at a basic level, then becomes a critical undertaking. Two well characterized schizophrenia susceptibility genes that have emerged are neurotrophic factor, Neuregulin-1 (NRG1), and its receptor ErbB4 (Stefansson et al., 2002; Yang, 2003; Mei and Xiong, 2008; Mei and Nave, 2014).
27
1.3.2 Neuregulin-1 and ErbB4
NRG1 is a trophic factor belonging to a family of proteins containing an EGF-like domain (Mei and Xiong, 2008; Mei and Nave, 2014). Its function is mediated by
ErbB proteins, with ErbB4 being the only autonomous receptor (Guy et al., 1994;
Sliwkowski et al., 1994 ; Garrett et al., 2003). ErbB4 is a single transmembrane protein that serves as a receptor tyrosine kinase receiving several growth factors as ligands, NRG1 being the most well characterized (Mei and Xiong, 2008; Mei and Nave, 2014).
1.3.2.1 History of Growth Factors and Their Receptors
Growth factors’ discovery owes its origins to observation from Victor
Hamburger’s lab that when limb buds are amputated from chicken embryos, the nerves that normally enervate them retract and die and that when additional limb buds are implanted, they are innervated by nerve fibers. Additionally, mouse sarcoma tumors were also innervated when transplanted to these chicken embryos. When Rita Levi-Montalcini repeated these experiments and instead implanted the sarcoma tumor in a place where nerve cells usually don’t grow and obtained the same results, she was led to the hypothesis that these cells must be secreting a factor that promotes nerve growth (Levi-Montalcini, 1952). Stanley
Cohen was later recruited to the team, ground these cells up and found that the extracts themselves promoted nerve growth (Cohen and Levi-Montalcini, 1957).
This landmark discovery and isolation of nerve growth factor (NGF) in 1957 and
28 subsequent work would earn Cohen and Levi-Montalcini Nobel prizes nearly 30 years later.
They later discovered that an extract from salivary glands was an even more potent potentiator of nerve growth, but the extract appeared to have a second component that also promoted early eye opening and teeth eruption when injected into mouse pups (Cohen, 1962). This second component ended up being epidermal growth factor (EGF). EGF was found to be a potent stimulator of proliferation of cultured human fibroblast and thus they were thought to contain a receptor for EGF. Radiolabeled EGF indeed did bind to the surface of these and other cells, and only un-labeled EGF could block this effect.
Interestingly, many proteins were phosphorylated, specifically on tyrosine residues, in response to EGF treatment, including a 170 kDa protein that turned out to be the receptor itself, EGF receptor (EGFR or ErbB1) (Cohen and
Carpenter, 1975; Hunter and Cooper, 1981). Separate efforts by virologists found that an avian erythroblastosis virus, which was known to induce abnormal presence of nucleated red blood cells (erythroblast) and sarcomas (a type of cancer), encoded two proteins, ErbA and ErbB, and that ErbB was critical for the viruses’ oncogenic effect (Yamamoto et al., 1983). When EGFR was later sequenced, it was found to be homologous to ErbB whose sequence had already been published (Downward et al., 1984). This was the first ties of EGFR to cancer and henceforth was found to be overexpressed in many types of cancers including glioblastomas (Hendler and Ozanne, 1984; Libermann et al., 1985;
Sugawa et al., 1990).
29
ErbB2, sometimes called human EGFR2 (HER2), was discovered by
Robert Weinberg who was specifically looking for carcinogen-induced mutations that led to tumors. When pregnant rats were exposed to a certain carcinogen, their offspring would often have brain tumors. When the affected gene, originally termed Neu, was sequenced it had surprising homology to EGFR (Schechter et al., 1984; Yamamoto et al., 1986). Efforts to find similar mutations in ErbB2 in human cancers were fruitless but like EGFR, ErbB2 was found to be over expressed in many cancers (Kraus et al., 1987; Slamon et al., 1989). With the discovery of a second EGF-like receptor, it was postulated that there would be an
EGF-like growth factor that could bind and activate ErbB2. Two groups isolated this putative factor calling it Heregulin and Neu differentiation factor respectively
(Holmes et al., 1992; Wen et al., 1992). Although interestingly it could activate
ErbB2, it did not directly interact with it. Almost simultaneously, a third group isolated a factor that stimulated Schwann cell proliferation, calling it Glial growth factor (Goodearl et al., 1993; Marchionni et al., 1993), and a fourth group isolated a factor that was thought to stimulate acetylcholine receptor clustering at the neural muscular junction, calling it Acetylcholine receptor inducing activity (ARIA)
(Corfas et al., 1993; Falls et al., 1993). The factors isolated by the four groups were all encoded by one gene that is known as NRG1 today.
To this day, ErbB2 remains an orphan receptor as no known ligands bind to it. While, it was later revealed that EGF could stimulate ErbB2 to heterodimerize with EGFR, none of the NRGs were shown to bind EGFR (Kokai et al., 1989; Goldman et al., 1990). This left open the idea that other ErbB
30 receptors were yet to be found. ErbB3 and ErbB4 were soon discovered and found to bind NRG1 (Kraus et al., 1989; Plowman et al., 1993). Intriguingly,
ErbB3 while binding NRG1, was not activated. Apparently, a few unconserved residues on the kinase domain left this receptor kinase dead or with little kinase activity. This meant that of all the ErbBs only ErbB4 is an autonomous receptor for NRG1.
1.3.2.2 Structure and Function
NRG1 belongs to a family of growth factors that to date include six members, NRG1-6, all containing an EGF-like domain, NRG1 being the most well characterized (Mei and Nave, 2014). NRG1 is 1.125 Mb gene with 21 exons located on human chromosome 8p12 and mouse chromosome 8 (Steinthorsdottir et al., 2004). Although there are greater than 30 possible isoforms the gene is known to code for 6 unique proteins, NRG1 type I-VI, all with type specific N- terminal sequences (Falls, 2003; Mei and Xiong, 2008). The proteins are produced as pro-NRG1s associated with the cell membrane via a transmembrane domain. The EGF-like domain is proximal to the membrane on the extracellular side (Figure 4A).
31
I II III IV V VI
(Mei and Xiong, 2008)
Figure 4. Neuregulin-1 Isoforms, ErbBs and ErbB4 Isoforms.
(A) Diagram of domains of NRG1 and common isoforms (Mei and Xiong, 2008).
NRG1 exist as pro-NRG1, and then is cleaved by a metalloprotease to form mature NRG1. Type I, II, and IV-VI are secreted after being cleaved whereas type III remains membrane bound. Types I, II, IV and V have an Ig domain. All isoforms contain an EGF-like domain. ( B) ErbB receptors, EGFR (ErbB1), ErbB2,
ErbB3 and ErbB4. EGFR, ErbB3 and ErbB4 can exist in either a closed or open state. ErbB2 is constitutively open. ( C) Diagram of domains of ErbB4 and different isoforms. CRD, cysteine rich domain; Ig, immunoglobulin; pYn, pyrimidine-nucleoside phosphorylase; TM, transmembrane.
33
Types I, II, IV, and V contain an immunoglobulin-like (Ig) domain between their
EGF-like domain and type specific sequence and are sometimes collectively referred to as Ig-NRG1. Type III is unique, in that it contains a second transmembrane domain, such that while all other types are secreted after they undergo extracellular cleavage, it remains membrane bound. Its second transmembrane domain is rich in cysteine residues and is sometimes referred to as cysteine rich domain (CRD)-NRG1 (Figure 4A) (Falls, 2003; Mei and Xiong,
2008). In a study of mRNA expression in postmortem brain, type III was most abundant followed by type II in both young and aged individuals (Liu et al., 2011).
NRG1 mediates its functions through ErbB receptors.
ErbB receptors are a family of single pass transmembrane proteins that are receptor tyrosine kinases and include EGFR, ErbB2, ErbB3 and ErbB4
(Figure 4B). Members of the ErbB family of receptors can dimerize with any other member, making four possible homodimers and six possible heterodimers. In order to form dimers, ErbB receptors must bind a ligand. This induces a conformational change in the extracellular domain, changing it from a closed state to an open one. However, ErbB2 is in a constitutively open state (Figure
4B) (Cho et al., 2003). Its sole purpose appears to be a partner with other ErbBs, possibly to lower the threshold of ligand necessary for signaling events. It is not thought to form homodimers under normal conditions; however, it can under certain in-vitro conditions and may do so aberrantly in-vivo contributing to its potent oncogenic properties (Canbay, 2003; Gerber et al., 2004). ErbB3 on the other hand is able to bind ligands, NRG1, 2, and 6 but has little to no intrinsic
34 kinase activity and thus relies on other ErbBs to become activated (Cho and
Leahy, 2002). EGFR can form functional homodimers, but does not bind any of the NRGs, and instead has its own list of ligands some of which overlap with that of ErbB4 (Linggi and Carpenter, 2006). This leaves ErbB4 as the only autonomous NRG1 receptor, although it can also signal through NRGs 2-5, and
EGFR shared ligands, betacellulin (BTC), heparin-binding EGF (HB-EGF), and epiregulin (EPR) (Hobbs et al., 2002; Mei and Nave, 2014).
ErbB4 is a 1.5 Mb gene located on human chromosome 2q33.3-34 and mouse chromosome 1 with 29 exons that codes four distinct isoforms of the protein (Carpenter, 2003; Mei and Xiong, 2008). At the N terminal is a signal sequence followed by four extra cellular domains and a stalk region that can exist in two different isoforms, juxtamembrane domain (JM)-a, and JMb. JMa gives
ErbB4 a slightly longer stalk and makes it more susceptible to extracellular cleavage (Elenius et al., 1997). Following the transmembrane domain on the intracellular side, there is a kinase domain, and one of two isoforms, cytoplasmic domain (CYT)-1 or CYT2. CYT1 contains a binding site for phosphatidylinositol-3 kinase (PI3K) that is missing in CYT2 (Elenius et al., 1999). Finally, at the carboxyl terminal there is a post synaptic density (PSD) binding motif (Figure 4C)
(Garcia et al., 2000; Huang et al., 2000).
Canonical NRG1-ErbB4 signaling begins with pro-NRG1 being cleaved extracellularly from the signaling cell by metalloproteases like tumor necrosis factor α converting enzyme (TACE / ADAM17) (Montero et al., 2000), β-site of amyloid precursor protein cleaving enzyme 1 (BACE1) (Hu et al., 2006), or
35
Meltrin-β (ADAM19) (Yokozeki et al., 2007). Soluble NRG1 can then bind ErbB3 or ErbB4 on the target cell (Figure 5A). Binding NRG1 induces a conformational change in the extracellular region of the ErbB receptor shifting it from a closed state to an open one. The ErbB receptor can then bind another ligand bound
ErbB or ErbB2. When ErbB4 forms homo or heterodimers, the dimer auto- phosphorylates. Several adapter proteins can then interact with the phosphorylated dimer. For instance, SHC (Culouscou et al., 1995), signal transducers and activators of transcription (STAT)3 and 5 (Olayioye et al., 1999), growth factor receptor bound protein (GRB)2 and 7 (Sepp-Lorenzino et al.,
1996), and just another or Janus kinase (JAK) (Carpenter, 2003; Mei and Xiong,
2008). SHC and GRB2 promote Ras-Raf-MEK-ERK pathway signal transduction.
ERK can then regulate several transcription factors (Figure 5A). In peripheral cells this pathway is associated with mitogenesis. CYT1 isoform of ErbB4 has the addition of a PI3K binding site. PI3K-AKT-mTOR signal transduction is another common pathway activated by NRG1-ErbB4 signaling (Elenius et al., 1999). In the brain, it appears that NRG1-ErbB4 signaling may involve endocytosis of
ErbB4 (Yang et al., 2005; Liu et al., 2007).
36
(Mei and Nave, 2014)
Figure 5. Neuregulin-1 – ErbB4 signaling.
(A) Canonical NRG1-ErbB4 signaling. ErbB4 shares ligands with EGFR: betacellulin (BTC), heparin-binding EGF (HB-EGF), and epiregulin (EPR); and with ErbB3: NRG1 types I-VI, and NRG2; as well as having ligands unique to
ErbB4: NRG3, NRG4 and NRG5 (Mei and Nave, 2014). Pro-NRG1 is cleaved extracellularly by BACE1 and further processed by neuropsin. The EGF-like domain then binds to ErbB4 inducing a conformational change from a closed state to an open one. ErbB4 can then dimerize with another ErbB. The ErbB dimer autophosphorylates allowing the binding of adapter proteins like Shc, Grb2 and PI3K. Common signaling cascades activated are Ras-Raf-MEK-Erk and
PI3k-Akt-mTOR. These signaling cascades can culminate with transcriptional regulation. ( B) Non-canonical NRG1-ErbB4 signaling. NRG1 can be cleaved intracellularly by γ-secretase for nuclear signaling (Mei and Nave, 2014). ErbB4 can be cleaved extracellularly by BACE1. This could bind free NRG1 in the extracellular space to prevent additional signaling or to membrane bound NRG1 for backward signaling. Additionally, ErbB4 may bind type III NRG1 either in free form or through cell-cell contact for transmembrane dependent signaling. ErbB4 can be cleaved intracellularly by γ-secretase for nuclear signaling.
38
In addition to the commonly activated signaling pathways during NRG1-
ErbB4 signaling, there are also less common mechanisms for NRG1 and ErbB4 to signal to each other. JMa isoform of ErbB4 has a juxtamembrane domain that is susceptible to extracellular cleavage resulting in ecto-ErbB4 (Elenius et al.,
1997). This may serve as a way to bind soluble NRG1 in extracellular space to inhibit additional canonical signaling. It has been demonstrated that ecto-ErbB4 acts as a ligand for NRG1 in a backward signaling process (Bao et al., 2003).
This type of signaling may be significant for type III NRG1, which stays anchored to the cellular membrane after cleavage. Both NRG1 and ErbB4 can additionally undergo intracellular cleavage by γ-secretase leading to nuclear signaling events that may regulate transcriptional events (Figure 5B) (Ni et al., 2001; Bao et al.,
2003; Sardi et al., 2006).
1.3.2.3 Function in the Brain
During neurodevelopment, glutamatergic neurons are born in the subventricular zone (SVZ) and then migrate radially. However, GABAergic interneurons are born in medial, lateral, and caudal ganglionic eminences (MGE,
LGE, and CGE) and instead migrate tangentially (Figure 6A) (Marin et al., 2010).
ErbB4 transcripts are seen in the MGE at embryonic day (E)14 in mice, a time when interneurons are beginning to migrate (Yau et al., 2003). Although it is debated whether NRG1 serves as a chemoattractive or chemorepulsion cue, it is agreed upon that NRG1-ErbB4 signaling is important for interneuron migration
(Flames et al., 2004; Li et al., 2012a). The two reports agreed that genetic
39 deletion of ErbB4 led to a decrease in GABAergic interneurons in cortical areas after a period when migration should be complete. One study also implicated
NRG1-ErbB4 signaling in migration of neuroblasts along the rostral migratory stream in the adult rat (Anton et al., 2004). Neuroblasts are born in the SVZ in the adult and migrate to the olfactory bulb, where they become olfactory interneurons. In this study, it was demonstrated that ErbB4 transcripts were highly expressed along the rostral migratory stream and that conditional deletion of ErbB4 in neurons led to reductions in interneurons in the adult olfactory bulb.
They further showed that while wild-type explants from SVZ preferentially moved toward cells transfected with NRG1 type III, explants from ErbB4 deleted mice did not (Anton et al., 2004).
Earlier in-vitro studies implicated NRG1-ErbB signaling in radial migration of excitatory neurons and suggested that ErbBs, including ErbB4, may be expressed in radial glia (Anton et al., 1997; Rio et al., 1997). However, later in- vivo studies using various conditional deletions of NRG1 (Brinkmann et al., 2008) or ErbB2 and /or ErbB4 (Pitcher et al., 2008; Barros et al., 2009) could not confirm these results. Treatment of NRG1 stimulated neurite outgrowth in cultured hippocampal or cortical neurons that are mostly glutamatergic (Gerecke et al., 2004; Audisio et al., 2012). Others dispute this claiming that this effect is only observed in GABAergic interneurons (Krivosheya et al., 2008; Cahill et al.,
2012). The latter view would seem to be supported by in-vivo characterization of mice lacking ErbB4 as they did not show any abnormalities in dendritic arborization (Barros et al., 2009).
40
(Mei and Nave, 2014)
Figure 6. Neuregulin-1 – ErbB4 Signaling in the Brain.
(A) NRG1-ErbB4 signaling has been implicated in interneurons (blue) migration from the MGE to cortical areas, axon and dendritic development, and synaptogenesis. Glutamatergic neurons (orange) migrating from ventricular zone
(VZ) along radial glia (green). (B) Hippocampal circuitry, boxed area enlarged.
Boxed areas a, b, c, and d, enlarged in Ca , b, c, and d respectively. (Ca )
Excitatory-inhibitory synapse. Transmembrane or soluble NRG1 may interact with ErbB4 to promote postsynaptic or dendritic development. NRG1-ErbB4 backward signaling may promote presynaptic development. ( Cb and c) Similar interactions may occur on either inhibitory-excitatory or inhibitory-inhibitory synapses. ( Cd ) NRG1 interacts with ErbB4 either at the soma or synapse to promote GABA release to inhibit Glutamatergic neuron firing and regulate LTP.
41
Mice lacking ErbB4 in either all neurons or specifically in GABAergic neurons did show decreased numbers and size of spines on excitatory neurons suggesting a synaptic deficit (Barros et al., 2009; Del Pino et al., 2013; Yin et al.,
2013a). This also suggests that the spine deficit may be through backward signaling (Figure 6B and Ca). NRG1-ErbB4 signaling appears to be important for
GABAergic synaptogenesis as well. Treatment of cultured cortical neurons with
NRG1 could increase the number of excitatory synapses on GABAergic, but not glutamatergic neurons, whereas treatment with ecto-ErbB4 could decrease excitatory synapses in GABAergic neurons (Ting et al., 2011). Genetic ablation of
ErbB4 in interneurons could reduce the number of inhibitory synapses on excitatory neurons (Figure 6Cb) (Fazzari et al., 2010).
NRG1-ErbB signaling is an important component of axon guidance and myelination. In the periphery, ErbB2 and ErbB3 on Schwann cells are thought to mediate these functions (Carroll et al., 1997; Riethmacher et al., 1997; Garratt et al., 2000; Brinkmann et al., 2008), whereas in the central nervous system, ErbB3 and ErbB4 on oligodendrocytes are believed to be the main players (Vartanian et al., 1997). Halpo-deficiency of type III NRG1 was reported to lead to mild hypomyelination phenotypes in the brain (Taveggia et al., 2008). Over expression of a dominant negative form of ErbB4 in oligodendrocytes led to a hypomeyelination phenotype, as well (Roy et al., 2007). Over expression of type
III NRG1 was reported to cause hypermyelination (Brinkmann et al., 2008).
However, this report also reported that knock out of NRG1 using various CNS
42 specific Cres did not lead to any obvious myelination defects nor did deletion of
ErbB4 (Brinkmann et al., 2008). Loss of ErbB3 did appear to lead to severe hypomyelination (Makinodan et al., 2012) and severe laminar defects in the cerebellum (Sathyamurthy et al., 2015).
Among the ErbBs, ErbB4 is the only one that is continuously expressed into adulthood at high levels (Fox and Kornblum, 2005). This suggests that besides NRG1-ErbB4 signaling’s role in development, it may play a functional role in adulthood. It has been demonstrated that NRG1 treatment can enhance the release of GABA (Figure 6Cd) (Woo et al., 2007; Chen et al., 2010; Li et al.,
2012b) and dopamine (Yurek et al., 2004; Kwon et al., 2008). The enhanced
GABA release could be blocked by either pretreatment with ecto-ErbB4 or with genetic ablation of ErbB4 in PV-positive GABAergic neurons (Woo et al., 2007;
Chen et al., 2010; Li et al., 2012b).
The mechanism behind NRG1 enhanced neurotransmitter release is not well understood, but several models have been proposed. In one model, voltage gated potassium channel Kv1.1 was invoked as the mechanism (Li et al., 2012b).
Voltage gated potassium channels are important for repolarizing neurons after an action potential is fired. In this study, the authors demonstrated that NRG1 treatment could phosphorylate Kv1.1. Treatment with ecto-ErbB4 could increase potassium currents and specific blockade of Kv1.1 could abolish this effect (Li et al., 2012b). Another model posits that NRG2-ErbB4 signaling can promote endocytosis of GABA receptors, thus disinhibiting the interneurons expressing
ErbB4 (Mitchell et al., 2013). However, another report suggested that NRG1
43 treatment could decrease sodium currents, thus leading to less excitability
(Janssen et al., 2012).
NRG1 treatment appears to have an opposing role in glutamatergic neurons, inhibiting their firing. Treatment of NRG1 could either suppress long term potentiation (LTP) in CA1 hippocampal neurons (Huang et al., 2000; Pitcher et al., 2008) or reverse it (Kwon et al., 2005). Several models have been proposed to explain this phenomenon. When ErbB4 is deleted from GABAergic interneurons, NRG1 treatment is no longer able to modify LTP or pyramidal neuron firing (Chen et al., 2010; Wen et al., 2010; Li et al., 2012b; Tan et al.,
2012). Thus it appears that NRG1’s ability to enhance GABA transmission may indirectly suppress glutamatergic neuron firing (Figure 6Cd). Other models have been postulated. In one, NRG1-ErbB4 signaling is thought to suppress Src kinase’s ability to upregulate NMDA receptors (Pitcher et al., 2011). This view is controversial, as NRG1-ErbB signaling has long been thought to activate Src and related kinases (Olayioye et al., 1999; Bjarnadottir et al., 2007; Cahill et al.,
2012). A third model suggests that NRG1’s ability to regulate LTP is mediated through stimulating dopamine release and that the D4 receptor is critical for this process (Kwon et al., 2008). The mechanism behind this model is not entirely clear, as dopamine release in the hippocampus has long been implicated in the promotion of LTP, not the suppression of it, although this is through D1 type receptors and not D4, a D2 type receptor (Figure 1) (Huang and Kandel, 1995;
Bach et al., 1999; Kellendonk et al., 2006).
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1.3.2.4 Neuregulin-1 and ErbB4 Association with Schizophrenia
With all the roles NRG1-ErbB4 signaling plays in neurodevelopment and the additional functional roles it plays, it should come as no surprise that both
NRG1 and ErbB4 have been implicated in the psychiatric disorder, schizophrenia
(Mei and Xiong, 2008; Mei and Nave, 2014). NRG1 was originally linked and associated with schizophrenia in an Icelandic population in 2002 (Stefansson et al., 2002) and almost simultaneously in a Han Chinese population (Yang, 2003).
In the Icelandic study, a genome wide linkage scan was first conducted on 105 patients from 33 families. A region on chromosome 8p12-21 emerged with the highest log of odds ratio (LOD) 3.06 for linkage disequilibrium (Figure 3). A LOD of 3 is considered to be a 1 in 1000 chance of being non-randomly associated
[log (1000) = 3]. Subsequent fine mapping revealed NRG1 as the gene of interest at 8p12.
After picking up additional patients, several haplotypes emerged in this region with a large overlapping segment shared. When compared to the general population in Iceland, affected individuals had this core haplotype 15.4% of the time compared to 7.4% in unaffected individuals. Having the haplotype appeared to confer a 2.2x risk for having schizophrenia. The core haplotype included a non-synonymous single nucleotide polymorphism (SNP) on exon 1 that effects type II NRG1 (Stefansson et al., 2002). Recall type II is the second most expressed isoform, only behind type III, that remains membrane bound (Liu et al.,
2011). The study from the Han Chinese population identified this same SNP as well as two others (Yang, 2003). Since the initial two reports associating NRG1
45 as a schizophrenia risk gene, numerous reports from multiple populations have supported this notion, including Scottish (Stefansson H, 2003), Portuguese
(Petryshen et al., 2005), Irish (Norton et al., 2006), Norwegian (Athanasiu et al.,
2010), and American (Sullivan et al., 2008; Shi et al., 2009). Additional support comes through the rigors of meta-analyses (Li et al., 2006; Munafo et al., 2006;
Agim et al., 2013) and genome-wide association studies (GWAS) (Sullivan et al.,
2008; Shi et al., 2009; Athanasiu et al., 2010).
More than 23,000 SNPs have been identified on NRG1 , of which around
40 have been associated with schizophrenia thus far. Of these, only 4 are non- synonymous exonal SNPs: SNP8NRG433E1006 on type II specific sequence,
Arginine to Glycine; rs3924999 on the Ig domain, Arginine to Glutamine; rs10503929 on the stalk region, Valine to Leucine; and rs74942016 on the transmembrane domain, Methionine to Threonine (Mei and Nave, 2014).
Through phenotype based studies, these SNPs have been associated with reduced prepulse inhibition (PPI), a measure of sensory motor gating and with cognitive deficits (Lin et al., 2005; Hong et al., 2008; Roussos et al., 2011). Most
SNPs then are either in intronic regions or in the 5’ untranslated region. This has led to the hypothesis that these may contribute to altered expression levels of
NRG1 through affecting the promoter, enhancers, or sites of epigenetic modification.
Indeed, altered expression of NRG1 has been observed in schizophrenics in several studies. Transcripts of type I NRG1 were reported to be higher in the dorsal lateral PFC (DLPFC) or hippocampus in schizophrenics compared to
46 controls (Hashimoto et al., 2004; Law et al., 2006). However, another study reported that type I transcripts were lower in medial PFC, while type II were elevated (Parlapani et al., 2010). Besides mRNA, NRG1 protein was reported to be higher in a schizophrenic population (Chong et al., 2008). While many studies have not found connections between the disorder of schizophrenia and NRG1 expression levels, they have linked specific SNPs to altered expression.
SNP8NRG221132 was associated with the increase in type I (Law et al., 2006).
In one report, rs7014762 was associated with decreased expression of type III
NRG1 (Nicodemus et al., 2009), while in another, it was associated with increased expression (Weickert et al., 2012). SNP8NRG243177 was related with increased expression of type IV (Law et al., 2006; Moon et al., 2011). Therefore,
NRG1 may be increased in schizophrenia and most, but not all reports suggest that many SNPs are associated with increased expression of various isoforms.
SNPs on NRG1 have shown relations with brain abnormalities and behavioral deficits common to schizophrenia. SNP8NRG243177 has been associated with decreases in both grey and white matter volumes, structural connectivity, as well as, with increased ventricle volume (McIntosh et al., 2008;
Mata et al., 2009; Barnes et al., 2012; Cannon et al., 2012). SNP8NRG221533 was linked with decreased integrity of white matter (Winterer et al., 2008).
SNP8NRG243177 was also connected to lower frontal and temporal lobe activity during a sentence completion task, with psychotic symptoms, lower intelligence
(IQ) (Hall et al., 2006), and reduced PPI (Hong et al., 2008; Roussos et al.,
2011). It also appears to be related to a reduced working memory capacity in
47 non-schizophrenic carriers (Stefanis et al., 2007). Non-schizophrenic carriers of
SNP8NRG221533 appeared to have decreased verbal fluency (Kircher et al.,
2009), and deficits in smooth eye pursuit (Schmechtig et al., 2010). Interestingly, one report suggested, carries of SNP8NRG243177, who are intellectually gifted, are more creative when compared to non-carriers of similar IQ (Keri, 2009), providing a possible mechanism as to why certain SNPs are not evolutionarily selected against.
ErbB4 has also been associated with schizophrenia. In 2006 three separate SNPs on ErbB4 were found to be associated with schizophrenia in an
Ashkenazi Jewish population (Silberberg et al., 2006). Since the initial report
ErbB4 has been associated with schizophrenia in several other populations including: American (Nicodemus et al., 2006; Walsh et al., 2008; Shi et al., 2009);
Han Chinese (Lu et al., 2010); and European (Agim et al., 2013). Like the association of NRG1 , ErbB4 survives the rigors of GWAS (Shi et al., 2009) and meta-analysis (Agim et al., 2013).
Both transcripts and protein of ErbB4 have been reported to be higher in the PFC of schizophrenics (Chong et al., 2008; Joshi et al., 2014). Isoform JMa;
CYT1 was reported to be elevated in the DLPFC of schizophrenics (Silberberg et al., 2006; Law et al., 2007) and that a haplotype containing the three SNPs originally identified (rs7598440, rs707284, rs839523) was connected to higher expression of JMa; CYT1 in both hippocampus and DLPFC (Law et al., 2007).
SNP rs7598440 specifically, was correlated with higher concentrations of GABA in a spectroscopy study in healthy controls (Marenco et al., 2011). One study,
48 while failing to find any differences in expression of either NRG1 or ErbB4, found that postmortem brain tissue incubated with NRG1 had increased levels of phosphorylated ErbB4 in schizophrenics (Hahn et al., 2006). Together these results suggest a gain of function in ErbB4 signaling in schizophrenia pathology.
However, a rare deletion of ErbB4 ’s kinase domain was also seen in a schizophrenic patient (Walsh et al., 2008), and SNP rs4673628 on ErbB4 has been linked to reduction in white matter integrity (Zuliani et al., 2011). These suggest that either gain or loss of function in ErbB4 signaling could play a role in schizophrenia.
Another promising schizophrenia risk gene that has been identified is
NRG3 , a ligand specifically for ErbB4. SNPs rs10883866, rs10748842, and rs6584400 on NRG3 were associated with schizophrenia (Kao et al., 2010) and showed correlation to positive symptoms in schizophrenic patients (Chen et al.,
2009). SNP rs6584400 additionally predicted attention deficits in both schizophrenics and bi-polar patients (Meier et al., 2013). SNP rs10748842 was connected to decreased DLPFC activation during a working memory task in patients (Tost et al., 2014). While another study suggested that rs6584400 and rs10883866 were related to better performance on an attention task in patients, but worse performance in healthy controls (Morar et al., 2011).
Complementing the work done in human populations, showing correlations between genes and disorders, brain abnormalities, and phenotypes is work done in genetically modified mice that can demonstrate causal effects of gene deletions. Mice that were haplo-deficient for NRG1 showed hyperactivity
49
(Gerlai et al., 2000; Stefansson et al., 2002; Boucher et al., 2007), that could be reversed with the atypical antipsychotic clozapine, deficits in PPI (Stefansson et al., 2002), and sensitivity the psychoactive chemical tetrahydrocannabinol (THC)
(Boucher et al., 2007). An EGF-like domain specific NRG1 haplo-deficiency also conferred hyperactivity and when challenged with NMDA antagonist MK-801, displayed PPI deficits (Duffy et al., 2008). These mice also displayed an increase in 5-HT2A receptors, suggesting a serotonin deficit (Dean et al., 2008). Type III specific haplo-deficient mice displayed short-term memory deficits and impaired
PPI, that could be reversed with nicotine treatment (Chen et al., 2008).
Intriguingly, overexpression of NRG1 could also cause many schizophrenia relevant behavioral phenotypes including hyperactivity, impaired working memory, learning and memory, social interaction, and PPI (Kato et al., 2010; Yin et al., 2013b). This suggests that NRG1 must be at an optimal level; too much or too little can lead to dysfunction.
More intriguing yet, these mice developed with normal levels of NRG1, and overexpression was turned on a few weeks before behavioral phenotyping.
Mice that developed with overexpression of NRG1 and then had the overexpression turned off just prior to testing did not display these altered behaviors (Yin et al., 2013b). These results suggest that the behavioral abnormalities are a result of NRG1-ErbB4 signaling role in brain function and not during development.
Halpo-deficiency of ErbB4 also produced hyperactivity and a mild deficit in
PPI in mice (Stefansson et al., 2002). Mice with a null deletion of ErbB4 (heart-
50 rescued) were hyperactive, had deficits in fear conditioned memory, and PPI, but showed reduced anxiety (Shamir et al., 2012). When ErbB4 was conditionally deleted specially from PV-positive interneurons, using a Cre-Lox system, mice continued to show many behavioral abnormalities including hyperactivity, impaired PPI (Wen et al., 2010; Shamir et al., 2012), that could be reversed by
GABA-A receptor agonist diazepam, impaired working memory (Wen et al.,
2010), and impaired fear conditioned memory (Chen et al., 2010). Interestingly, deletion of ErbB4 specifically from of PV-positive interneurons in the amygdala alone was sufficient to cause impaired fear conditioned memory, and reintroducing ErbB4 to cells in the amygdala in a global PV-positive ErbB4 knockout could partial rescue these impairments (Lu et al., 2014). Using a different Cre but targeting the same fast-spiking interneurons ( Lhx6::Cre ) produced many of the same behavioral phenotypes. The mice were hyperactive, had impaired working memory, impairments in social interaction, reduced PPI, but were less anxious (Del Pino et al., 2013). These mice additionally showed disruptions in gamma oscillations a phenotype that has been observed in schizophrenic patients (Kwon et al., 1999; Kissler et al., 2000).
A handful of reports have suggested that early life stress can lead to changes in NRG1 expression and that these correlate with other deficits. Social isolation for two weeks immediately following weaning led to dramatic loss of type III NRG1. This loss correlated with a hypomyelination phenotype and looked remarkably similar to a knockout of ErbB3 (Makinodan et al., 2012). Another report suggested that prenatal asphyxia led to decreased levels of NRG1
51 transcripts at 6 and 12 weeks postnatal in the medial PFC and increased COMT transcripts at 12 weeks. However, these changes did not affect their protein levels (Wakuda et al., 2015). Interestingly, transient exposure to NRG1 neonatally could lead to hyperdopaminergic states later in life. These mice additionally showed deficits in PPI, latent inhibition, social behaviors, and were sensitive to methamphetamine (Kato et al., 2011).
1.3.2.5 Controversies in Expression Profile
Despite all the roles NRG1-ErbB4 signaling plays in development, brain function in adults, and strong ties to schizophrenia, cellular targets of ErbB4 remain controversial. Many reports have placed ErbB4 in excitatory neurons
(Garcia et al., 2000; Huang et al., 2000; Ma et al., 2003; Kwon et al., 2005; Li et al., 2007; Iyengar and Mott, 2008; Barros et al., 2009; Pitcher et al., 2011). Some have suggested the ErbB4 regulates spines and synaptic plasticity via cell- autonomous mechanisms (Gu et al., 2005; Kwon et al., 2005; Li et al., 2007;
Pitcher et al., 2011). Others have disputed this view, demonstrating that ErbB4 on GABAergic neurons are sufficient to regulate spines of excitatory neurons (Yin et al., 2013a), or synaptic plasticity (Chen et al., 2010; Wen et al., 2010). ErbB4 transcripts were shown to be enriched in areas where interneurons are concentrated (Lai and Lemke, 1991; Woo et al., 2007) and its protein is expressed in GAD-positive hippocampal neurons (Huang et al., 2000; Woo et al.). ErbB4 is present in new born and migrating GABAergic interneurons (Yau et al., 2003) and in adult, in PV and somatostatin interneurons (Vullhorst et al.,
52
2009; Chen et al., 2010; Fazzari et al., 2010; Wen et al., 2010; Abe et al., 2011;
Neddens et al., 2011; Ting et al., 2011). Recent reports suggest that ErbB4 is exclusively expressed in GABAergic interneurons in cortex and hippocampus
(Vullhorst et al., 2009; Fazzari et al., 2010). ErbB4 mRNA was also detected in subcortical regions (Ozaki et al., 1997; Ma et al., 1999; Steiner et al., 1999;
Bruce et al., 2002; Anton et al., 2004); however, cellular targets remain unclear due to low resolution of earlier in-situ hybridization. Unfortunately, this question cannot be addressed by current antibodies against ErbB4 because of poor specificity and availability.
To identify neurons or cells where ErbB4 protein is expressed in the brain,
ErbB4-reporter mice were generated, where the red fluorescent protein tdTomato is expressed under the control of endogenous ErbB4 promoter. ErbB4-positive cells were characterized with different markers and in hybrid with GAD67::GFP mice. Results herein reveal that ErbB4 expression in cortex and hippocampus is restricted to GABAergic interneurons. In subcortical regions, ErbB4 is expressed in neurons or cells that express dopamine, serotonin, oxytocin, or S100β. These results identify novel cellular targets of ErbB4 and suggest a role of NRG1-ErbB4 signaling in monoamine transmission.
2. MATERIALS AND METHODS
All experimental procedures were reviewed and approved by the Institutional
Animal Care and Use Committee of Georgia Regents University. Mice were housed at 23 °C with a 12 hour light/dark cycle and food and water available ad libitum.
2.1 Generation of ErbB4-Reporter and ErbB4-Reporter ; GAD67::GFP Mice
Mice with targeted knock-in transgenes ErbB4::CreERT2 and Rosa::LSL- tdTomato (Madisen et al., 2010) were purchased from the Jackson Laboratory
(stock numbers 012360 and 007905, respectively). GAD67::GFP mice were a gift from Dr. Yuchio Yanagawa (National Defense Medical College Hospital,
Saitama, Japan) (Tamamaki et al., 2003). In ErbB4::CreERT2 mice, CreERT2 was inserted after the stop codon of the ErbB4 gene, following a ribosomal 2A skip (2A). CreERT2 expression is thus controlled by the promoter of the ErbB4 gene and had no effect of ErbB4 expression or function because CreERT2 is expressed as a separate protein (Madisen et al., 2010) (figure 7A). Rosa::LSL- tdTomato has a cassette inserted between exon 1(EX1) and exon 2 (EX2) of the
Rosa26 gene , which contains the CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid (CAG) promoter, a loxP-stop-loxP (LSL), tdTomato red florescent protein, and a woodchuck hepatitis virus post-translational regulatory
53
54 element (WPRE) (Madisen et al., 2010) (figure 7B). GAD67::GFP has an enhanced green florescent protein (eGFP) inserted after exon 1 of GAD67
(Tamamaki et al., 2003) (figure 7C).
Primers for genotyping polymerase chain reaction (PCR) are described as follows: ErbB4::CreERT2 : 5’-GGGAG GATTG GGAAG ACAAT-3’, 5’-CCTGC
AGGAA TACAG CACAA-3’, and 5’-AAAGA TGGGG CTCTT TGACA-3’;
Rosa: :LSL-tdTomato : 5’-AAGGG AGCTG CAGTG GAGTA-3’, 5’-CCGAA AATCT
GTGGG AAGTC-3’, 5’-GGCAT TAAAG CAGCG TATCC-3’, and 5’-CTGTT
CCTGT ACGGC ATGG-3’. GAD67::GFP : 5’-GGCAC AGCTC TCCCT TCTGT
TTGC-3’, 5,-GCTCT CCTTT CGCGT TCCGA CAG -3’, and 5’-CTGCT TGTCG
GCCAT GATAT AGACG -3’. PCR of ErbB4::CreERT2 resulted in a 465 base pair (bp) product for mice lacking the transgene, a 190 bp product for mice with the transgene and both products for mice with one allele of each. PCR of
Rosa::LSL-tdTomato resulted in a 350 bp product for mice lacking the transgene, a 200 bp product for mice with the transgene and both products for mice with one allele of each. PCR of GAD67::GFP resulted in a 654 bp product for mice lacking the transgene, and both a 265 bp and 654 bp product for mice heterozygotic for the transgene. Mice with two copies of GAD67::GFP are not viable and were thus were not utilized (figure 7D).
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Figure 7. Genotyping results for ErbB4-reporter and GAD67::GFP mice.
(A) Diagram of ErbB4 and ErbB4::CreERT2 genes and location of genotyping primers. ( B) Diagram of Rosa26 and Rosa26::LSL-tdTomato genes and location of genotyping primers. ( C) Diagram of GAD67 and GAD67::GFP genes and location of genotyping primers. ( D) Genotyping results for ErbB4-reporter and
GAD7::GFP mice respectively.
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Breeding ErbB4::CreERT2 and Rosa::LSL-tdTomato mice generated double transgenic ErbB4::CreERT2; Rosa::LSL-tdTomato mice (hereafter referred as ErbB4-reporter mice). P1 and P60 mice were administered with 180 mg/kg tamoxifen, once orally and intraperitoneal every other day over 5 days, respectively, and sacrificed at P7 and P70, respectively. Tamoxifen activates
CreERT2 in ErbB4 expressing cells and subsequent Cre-mediated removal of the stop signal in the LSL-tdTomato transgene. Ensuing tdTomato expression enabled detection of ErbB4-positive neurons. Five days following the final tamoxifen injection, mice were sacrificed and brains were harvested. All experiments were performed on male mice.
2.2 Neuron Culture
Neurons were prepared from embryonic day 18 (E18) ErbB4::CreERT2;
Rosa::LSL-tdTomato mouse embryos and cultured in neurobasal medium
(catalog #21103-049; Invitrogen) supplemented with B27 (catalog #17504-044;
Invitrogen), 600 μM L-glutamine (catalog #25-005-CI; Cellgro), and penicillin- streptomycin (catalog #30-003-CI; Cellgro). For low-density culture, 2.5 × 10 4 cells were seeded on a glass coverslip (1.8 cm in diameter; catalog #12-545-84;
Fisher Scientific) coated for 12 hours with 1 μg/ml poly-L-lysine (catalog #P2636;
Sigma) in 12-well plates (catalog # CLS3513-50EA, Sigma). At day in-vitro (DIV)
7, neurons were treated with 2 µM tamoxifen for 1-3 hours and fixed at DIV 12.
Neurons were fixed in 4% paraformaldehyde (PFA) at room temperature for 20 minutes. Neurons were washed 3 times in phosphate saline buffer (PBS)
57
(10 minutes each) and incubated (10 minutes) in 0.3% Triton/PBS. Next, neurons were incubated in 10% goat serum for 1 hour and with primary antibody for 24 hours at 4 ºC (Table 1). After washing 3 times in PBS (10 minutes each), neurons were incubated with Alexa Fluor 488 anti-mouse or rabbit antibody (Invitogen) for
1 hour at room temperature. Neurons were washed 3 times in PBS before being mounted on Superfrost Plus Microscope Slides (Fisher Scientific, Waltham, MA) and sealed with Vectasheild Mounting Media (Vector, Burlington, CA). Images were taken using a Zeiss LSM 710 scanning confocal microscope under a 40 X oil immersion objective.
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Table 1. Antibodies Used In the Study.
Species Antigen Clone Source Catalog # Dilution Mouse ErbB4 H4.77.16 Lab Vision, MA5-12888 1:300 Fremont, CA Rabbit ErbB4 Poly Gift (Zhu et al., 0618 1:500 1995) Rabbit PV Poly Swant, PV 25 1:1000 Switzerland Rabbit TH Poly Millipore, AB152 1:1000 Billerica, MA Rabbit 5-HT Poly Protos Biotech, NT-102 1:1000 New York, NY 5HTrab Rabbit Oxytocin Poly Millipore AB911 1:1000 Rabbit Vasopressin Poly Millipore PC234L 1:1000 Rabbit S100β Poly Dako, Z031129-2 1:500 Carpinteria, CA Mouse GFAP GA5 Millipore MAB360 1:500 Mouse MBP 1 Millipore MAB382 1:500 Rabbit Olig2 Poly Millipore AB9610 1:500 Rabbit NG Poly Millipore 07-425 1:1000 Rabbit DsRed Poly Clontech 632496 1:1000 Mountain View, CA
5-HT, serotonin; GFAP, glial fibrillary acidic protein; Olig2, oligodendrocyte transcription factor; PV, parvalbumin; MBP, myelin basic protein; NG, neurogranin; TH, tyrosine hydroxylase.
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2.3 Analysis of ErbB4-Positive Cells
After being anesthetized with a ketamine (100 mg/kg) / xylazine (20 mg/kg) cocktail, mice were transcardially perfused with PBS (2 ml / gram of body weight), followed by 4% PFA in PBS. Brains were harvested, incubated in 4%
PFA overnight, and dehydrated at 4 ºC in two steps with 15% and 30% sucrose in PBS. Brains were frozen in OCT (catalog # 14-373-65; Fisher) and sectioned into 25 µm slices on a cryostat microtome (Bosch Microm HM550, Stuttgart,
Germany) at – 20 ºC. Slices were collected serially in 24-well plates in a cryoprotective solution (30% propylene glycol, 30% glycerin, and 40% PBS) and stored at – 20 ºC until microscopy studies. Coronal brain slices from approximately 3.08 to 2.58 mm relative to bregma were used to access frontal association cortex, 0.74 to – 1.94 mm relative to bregma to access primary somatosensory cortex and – 1.34 to – 2.18 mm relative to bregma to access dorsal hippocampus.
Every 24 th slice (one from each 24 well plate), approximately 575 µm apart, was selected for analysis of ErbB4-positive cell density. Slices were first washed 3 times in PBS for 10 minutes each, mounted on Superfrost Plus
Microscope Slides (Fisher Scientific) and sealed with Vectasheild Mounting
Media (Vector) and Premium Cover Glass (Fisher Scientific). Images were taken on a Zeiss LSM 710 scanning confocal microscope with automated stage using a
20 X air objective and stitched together to make complete images of a brain region or section. Brain regions were identified by using anatomical atlases
60
(Paxinos and Franklin, 2001; Lein et al., 2007). For cortical and hippocampal slices, layers were discerned by differences in neuropil and with the aid of anatomical atlas (Lein et al., 2007).
To quantify the density of tdTomato-positive cells, a slice (25 µm in thickness) every 575 m m in a given region or nucleus was examined for positive cells. Positive cells were marked using imageJ software (NIH) multi point tool.
The cell density (cell number per mm 3) was calculated by the following equation: cell density = number of positive cells in n slices / sum volume of n slices. Brains were divided into 11 major brain regions, which were subdivided to 173 unique regions.
2.4 Immunofluorescent Labeling
Coronal sections from approximately – 0.58 to – 1.22 mm relative to bregma were used to access if ErbB4 was expressed in oxytocin or vasopressin neurons. Coronal sections from approximately – 3.08 mm to – 3.80 mm relative to bregma were used to determine if ErbB4 was expressed in dopamine neurons of the ventral tegmental area or substantia nigra compacta. Coronal sections from approximately – 3.80 mm to – 4.72 mm relative to bregma were used to reveal if ErbB4 was expressed in serotonin neurons of the raphe nuclei. Coronal sections from – 5.34 to – 5.52 mm relative to bregma were used to determine if
ErbB4 was expressed in norepinephrine neurons of the locus coeruleus. Coronal sections from approximately – 0.46 to – 6.64 mm relative to bregma were used to access the identity of the non-neuronal cell that expressed ErbB4.
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Brain slices were washed 3 times in PBS for 10 minutes each, blocked in a solution (10 % goat serum, 1 % BSA, 0.3 % Triton) for 1 hour at room temperature, and incubated with primary antibodies (Table 1) for 24 hours at 4
ºC. After washing 3 times in PBS for 10 minutes each, slices were incubated with
Alexa Fluor 488 or 405 anti-rabbit or mouse antibodies (Invitogen) for 1 hour at room temperature. Slices were washed 3 times in PBS for 10 minutes each, mounted on Superfrost Plus Microscope Slides (Fisher Scientific) with
Vectasheild Mounting Media (Vector) and covered with Premium Cover Glass
(Fisher Scientific). Images were taken on Zeiss LSM 710 scanning confocal microscope with automated stage using a 40 X oil immersion objective. They were stitched together for complete view of regions of interest.
2.5 Microdialysis
Male C57BL/6J mice were purchased from Jackson laboratories stock number 000664 for microdialysis experiments. Mice were anesthetized with a ketamine (100 mg/kg) / xylazine (20 mg/kg) cocktail. They were placed on a stereotaxic instrument (David Kopf Instruments, Tujunga, CA). Hair was removed from the scalp and an incision (~ 1.0 cm) was made along the midline. A 0.8 mm hole was drilled into the skull, 1.8-1.5 mm from bregma, and 0.5 mm from midline. A guide cannula (CMA 7, Harvard Apparatus) was lowered 1.5 mm into prelimbic and infralimbic cortices. The guide cannula was secured with dental cement. After dental cement cured, the incision was sutured with absorbable nylon sutures. The mice were then placed back in their home cage and left to
62 recover for 3 days. One day before the experiment, mice were again anesthetized. Dummy probes were removed from guide cannulas, 1 mm microdialysis probes were inserted (CMA 7), and mice were left to recover overnight. Artificial cerebral spinal fluid (ACSF) (150 mM NaCl; 3 mM KCl; 1.5 mM CaCl 2; 1 mM MgCl 2) was perfused at a rate of 1 µl/min using a syringe pump
(CMA 402). Samples were collected every 20 min. in 300 µl plastic vials in a refrigerated microfraction collecter (CMA 470) stabilized with buffering solution
(50 mM KH 2PO 4; 50 mg/ml EDTA; 3.38 mM phosphoric acid). Three samples were taking for a baseline reading of monoamines and then ACSF + NRG1 (3.33
µM) was perfused in for 20 min (~ 0.5 µg NRG1). Nine additional samples were then collected to follow the response.
2.6 High Performance Liquid Chromatography (HPLC)
Microdialysis samples were read on an EiCom (San Diego, CA) HPLC system: liquid chromatograph pump EP-700; column oven ATC-700; electrochemical detector ECD-700. Mobile phase composed of: 700 ml H 2O; 300 ml methanol; 164 µl acetic acid; 7.1 g Na 2SO4; 5.18 g acetate ammonium; 50 mg
EDTA, was pumped through the system at 250 µl/min. The column oven was set to 35º C, detection condition 750 mV vs. Ag/AgCl, with a time constant of 1.5 sec.
Samples were separated on an EiComPak CAX (cation exchange mode 2.0 x
200 mm) column, made to detect norepinephrine (NE), dopamine (DA), and serotonin (5-HT) without their metabolites in a single injection. NE, DA, and 5-HT analytic standards (Sigma-Aldrich) were used to calibrate the HPLC system
63 before each experiment (Figure 8A). Microdialysis probes were checked for recovery rate and determined to have ~ 10% recovery efficiency (Figure 8B).
Microdialysis samples were injected every 20 min. and chromatography were read on a computer. Amounts of NE, DA, and 5-HT were recorded for each time point. The three baseline samples were averaged for each mouse and used to standardize the remaining samples.
2.7 Statistical Analysis
Analysis of variance (ANOVA) was used to access variation of ErbB4- positive cell density across and within major brain regions. Regions were compared against an overall score post hoc using Bonferroni correction for multiple comparisons. For microdialysis experiments data were plotted against time for each neurotransmitter. Repeated measures ANOVA was used to determine if neurotransmitter levels changed over time. Linear trends were then accessed post hoc. Data were expressed as mean ± SEM. *, **, and *** indicate
P < 0.05, < 0.01, and < 0.001, respectively.
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Figure 8. Control Data for Microdialysis and HPLC.
(A) Chromatograph of standard samples 12.5 – 100 pg. ( B) Chromatograph of a standard sample 1000 pg (50 pg/µl x 20 µl) in black and amount dialysis probe was able to recover in 20 min at 1 µl/min from the same solution in red. NSp, non-specific.
3. RESULTS
3.1 Generation and Validation of ErbB4-Reporter Mice
Mice that expressed tdTomato red fluorescent protein specifically in ErbB4 expressing cells were generated by crossing ErbB4::CreERT2 mice, where
CreERT2 expression is under the control of endogenous ErbB4 promoter, with
Rosa::LSL-tdTomato mice (hereafter referred to as ErbB4-reporter mice)
(Madisen et al., 2010). CreERT2 is inactive in the absence of tamoxifen, and thus no tdTomato expression is expressed before. Mice were treated with tamoxifen, activating CreERT2 and thus mediating removal or the LSL cassette to enable tdTomato expression (Figure 9A). To determine whether tdTomato is faithfully expressed in cells that express ErbB4, cortical neurons from ErbB4-reporter mice were cultured at E18. Neurons were stained with 0618 antibody, a rabbit polyclonal antibody raised against the intracellular domain of ErbB4. This antibody has been used widely to label ErbB4 in cultured cortical neurons and in brain slices (Zhu et al., 1995; Garcia-Rivello et al., 2005; Ghashghaei et al.,
2006; Woo et al., 2007; Chen et al., 2010; Wen et al., 2010; Del Pino et al.,
2013). Cortical neurons expressing tdTomato were stained positive (Figure 9B).
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Figure 9. tdTomato Labels ErbB4-expressing Neurons.
(A) Schematic of transgenes ErbB4::CreERT2 and Rosa::LSL-tdTomato. A ribosomal 2A skip followed by an inducible Cre recombinase (Cre) estrogen receptor T2 (ERT2) was inserted after endogenous ErbB4 gene stop codon. A
CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid (CAG) promoter, a loxP (yellow triangles) flanked stop cassette, tdTomato red florescent protein and a woodchuck hepatitis virus post-translational regulatory element (WPRE) were inserted between exon 1(EX1) and exon 2 (EX2) of Rosa26 gene. After CreERT2 is activated by tamoxifen, the stop cassette is removed from Rosa::LSL- tdTomato resulting in Rosa::tdTomato and tdTomato will be expressed in all cells that were expressing ErbB4 at time of induction. tdTomato-positive neurons in culture ( B, C ) and in brain slices ( D, E ) of ErbB4-reporter mice reacted to polyclonal, 0618 ErbB4 antibody ( B, D, E ) and monoclonal NeoMarker-H4.77.16
ErbB4 antibody ( C). (F and G) Coronal sections of ErbB4-reporter mice brains, that was not induced with tamoxifen (F) and was induced with tamoxifen counter stained with PV in greenv( G). Arrows, neurons labeled by tdTomato and antibody; Arrow heads, neurons labeled by tdTomato alone.
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To further validate ErbB4-positive cells, cortical neurons were stained with another antibody, NeoMarker-H4.77.16, a mouse monoclonal antibody that was raised against the extracellular domain of ErbB4 and reacts with ErbB4- expressing neurons (Vullhorst et al., 2009; Neddens and Buonanno, 2011;
Neddens et al., 2011). The monoclonal antibody labeled more strongly tdTomato- expressing neurons than tdTomato negative neurons (Figure 9C). These observations demonstrate that tdTomato is expressed in neurons that express endogenous ErbB4 and can be used as a faithful indicator of ErbB4-positive neurons. This notion was supported by staining of cortical and hippocampal slices with the 0618 antibody (Figure 9D and E). ErbB4-reporter mice that were not induced with tamoxifen did not have any tdTomato-positive cells (Figure 9F compared to 9G).
3.2 Density of ErbB4-Positive Cells Varies Across Brain Regions
Global distribution of ErbB4-positive cells in the brain was first assessed.
ErbB4-positive cells were widely expressed, but showed obvious regional differences. Numerically, cortex had a higher concentration of positive cells than the overall average (Figure 10 and Table 2). Amygdala had a particular enrichment of ErbB4-positive cells with a significant difference from the overall total (Figure 10A, B and E). Thalamus, hindbrain, and cerebellum had significantly lower concentrations than that of overall brain (Figure 10E, G, and
H). Midbrain had a numerical lower number of ErbB4-positive cells than whole brain (Figure 10F and G). The density of ErbB4-positive cells in olfactory bulb,
69 basal ganglia, and hypothalamus was similar to the overall average for entire brain (Figure 10C, D and E).
Figure 10. Unique Distribution of ErbB4-positive Cells In Brain Regions.
Images (photographic negatives) of tdTomato florescence in ErbB4-reporter mouse brain slices illustrating overall ErbB4-positive cell expression. Composite images ( A) from a sagittal section 1.80 mm lateral from midline; ( B) from a sagittal section 2.52 mm lateral from midline; ( C) from a coronal section 4.28 mm relative to bregma; ( D) from a coronal section 1.54 mm relative to bregma; ( E) from a coronal section – 1.06 mm relative to bregma; ( F) from a coronal section –
3.80 mm relative to bregma; ( G) from a coronal section – 4.36 mm relative to bregma; ( H) and from a coronal section – 6.24 mm relative to bregma. Positions of the sagittal sections in A and B were indicated by line a, and line b in panels
C-H. Positions of the coronal sections in C-H were indicated by lines c-h in A and
B. Amyg, amygdala; BG, basal ganglia; Cb, cerebellum; Cx, cortex; HB, hindbrain; Hp, hippocampus; Hy, hypothalamus; MB, midbrain; OB, olfactory bulb.
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Table 2. Density of ErbB4-positive Cells In Different Brain Regions.
Brain regions ErbB4 cells / mm 3 Olfactory bulb 4600 ± 739 Cortex 7081 ± 780 Basal ganglia 3555 ± 607 Hippocampus 4667 ± 338 Amygdala 10149 ± 795 *** Thalamus 1245 ± 397 * Hypothalamus 5306 ± 1178 Midbrain 2271 ± 634 Hindbrain 1034 ± 270 * Cerebellum 1497 ± 469 * White matter 925 ± 199 ** Total 4379 ± 599
Values are expressed as mean ± SEM. F (10, 22) = 20.53, P < 0.001. N = 3 mice.
Bonferroni post hoc comparisons vs. total, * P < 0.05, ** P < 0.01, *** P < 0.001.
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It is known that LTP is difficult to induce in amygdala presumably because of high
GABAergic activity (Tang et al., 1999; Rammes et al., 2000; Sigurosson et al.,
2010). These results may provide an underlying mechanism, considering NRG1-
ErbB4 promotion of GABA release (Woo et al., 2007).
Because of recent immunolabeling reports demonstrating that ErbB4 is exclusively expressed in GABAergic interneurons in cortex and hippocampus
(Vullhorst et al., 2009; Neddens and Buonanno, 2011; Neddens et al., 2011), I began my studies with experiments to test this hypothesis. ErbB4-reporter mice were crossed with mice that expressed GFP under the endogenous promoter of
GAD67, which effectively labels all GABAergic cells (Tamamaki et al., 2003).
Unexpectedly, I identified many ErbB4-positive cells to be non-GABAergic in subcortical areas. I characterized these cells by immunohistochemical analysis using different antibody markers to elucidate their cell type. In the following, I describe the distribution of ErbB4-positive cells, cellular identity, and functional implications.
3.3 Olfactory System
The olfactory system, responsible for the sense of smell, can be divided into several sections: main olfactory bulb (MOB), accessary olfactory bulb, and olfactory cortical regions. The MOB has five characteristic layers (Figure 11A and
B): the glomerular layer, the most superficial, where axons of olfactory neurons form synapses onto dendrites of mitral cells in glomeruli; the outer plexiform layer containing axons and dendrites; the mitral layer, where the soma of mitral cells
73 are located; the internal plexiform layer with dendrites and axons; and the granule cell layer composed of granule interneurons (Scott, 2008; Nolte, 2009b).
ErbB4-positive cells were localized in all five layers; however, the density was highest in the glomerular layer, followed by the mitral cell layer (Table 3).
Moreover, ErbB4-positive cells in the granule cell layer appeared to project into the mitral cell layer, as well as, the outer plexiform layer (Figure 11B, arrows). In agreement, ErbB4 is present in migrating neuroblasts in the rostral migratory stream of the developing brain, which go on to populate the olfactory bulb (Anton et al., 2004; Ghashghaei et al., 2006; Long et al., 2007).
The olfactory bulb relays information to olfactory cortical regions like anterior olfactory nucleus, piriform cortex, olfactory tubercle, and dorsal tenia tectum. ErbB4-positive cells were present throughout the olfactory system (Table
4). Most, if not all, ErbB4-positive cells co-localized with GAD67-GFP, indicating they were GABAergic. However, many GAD67-positive cells did not show evidence of being ErbB4-positive, indicating that not all GABAergic neurons in the olfactory system express ErbB4 (Figure 11C and D).
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Figure 11. ErbB4-positive Cells Are Enriched In the Glomerulus and Mitral
Cell Layers of Olfactory Bulb.
(A) Diagram of mouse brain sagittal section (top) and coronal sections (middle, bottom). Dashed lines b and c of the sagittal section diagram indicate the position of coronal sections. ( B) Coronal section of ErbB4-reporter mouse brain, 4.28 mm relative to bregma. Area shown was indicated by the rectangle in the middle panel of A. ( C) Coronal section of ErbB4-reporter; GAD67::GFP mouse brain,
2.68 mm relative to bregma. Area shown was indicated by the rectangle in the bottom panel of A. ( D) Enlarged image from the boxed area in C. Arrows, neurons positive for ErbB4 and GAD67. Empty arrows, neurons positive for
GAD67 alone. 1, layer one; 2/3, layers two and three; 5, layer five; AON, anterior olfactory nucleus; DLO, dorsal lateral orbital cortex; FrA, frontal association cortex; gl, glomerulus layer; gr, granular layer; ipl, inner plexiform layer; LO, lateral orbital cortex; MO, medial orbital cortex; MOB, main olfactory bulb; ml, mitral layer; opl, outer plexiform layer; PrL, prelimbic cortex; VO, ventral orbital cortex.
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Table 3. ErbB4-positive Cells Are Enriched In Glomerular and Mitral Layers
In the Main Olfactory Bulb.
Main olfactory bulb layers ErbB4 cells / mm 3 Glomerular 16824 ± 3277 * Outer plexiform 1698 ± 409 Mitral 9304 ± 2358 Inner plexiform 5925 ± 1334 Granule cell 6232 ± 1472 Total 7467 ± 1583
Values are expressed as mean ± SEM. F (4, 25) = 7.76, P < 0.001. N = 3 mice.
Bonferroni post hoc comparisons vs. total, * P < 0.05.
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Table 4. Even Distribution of ErbB4-postive Cells In Olfactory System.
Olfactory areas ErbB4 cells / mm 3 Main olfactory bulb 5640 ± 1650 Accessory olfactory bulb 7221 ± 2146 Anterior olfactory nucleus 4546 ± 666 Olfactory tubercle 2713 ± 540 Piriform cortex 4428 ± 392 Taenia tecta 4830 ± 888 Dorsal peduncular 7069 ± 319 Total 4600 ± 739
Values are expressed as mean ± SEM. F (6, 14) = 1.94, P = 0.145. N = 3 mice.
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3.4 Cortex
The cerebral cortex is the outermost structure of the vertebrate brain, where high level processing occurs for varied processes including motor control and sensory perception. There appeared to be more ErbB4-positive cells in the cortex than other brain regions except the amygdala. However, the density of
ErbB4-positive cells varied in different cortical regions: with highest in parietal association, primary visual, and secondary visual cortices and lowest in frontal association, lateral orbital, and insular cortices (Table 5).
The cortex has a well-defined layered structure. I analyzed ErbB4-positive cell distribution in cortical layers of the primary somatosensory cortex (Figure
12A and B). Among the six layers, ErbB4-positive cells were densest in the external granular/external pyramidal layers (or layer 2/3), which is populated by pyramidal neurons projecting to other cortical areas. Following that were the molecular layer (or layer 1), where apical dendrites of pyramidal neurons are localized; the internal granular layer (or layer 4), where spiny stellate neurons are located; the internal pyramidal layer (or layer 5), which is populated with pyramidal neurons that project to the spinal cord and subcortical areas; and the polymorphic layer (or layer 6), which is composed of morphologically variable excitatory neurons that project to thalamus (Hendry, 2008; Nolte, 2009a). Similar distribution was observed in the frontal association cortex (Figure 11C).
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Table 5. Slight Variation In Density of ErbB4-positive Cells In Cortex.
Cortical regions ErbB4 cells / mm 3 Frontal association 5840 ± 237 Prelimbic 6341 ± 662 Infralimbic area 8695 ± 858 Medial orbital 6243 ± 520 Ventral orbital 6219 ± 764 Lateral orbital 5907 ± 1143 Dorsal lateral orbital 6001 ± 583 Cingulate 7518 ± 1297 Retrosplenial 7410 ± 1309 Parietal association 9429 ± 778 Temporal association 8392 ± 514 Primary motor 6462 ± 899 Secondary motor 6798 ± 932 Primary somatosensory 6747 ± 1105 Secondary somatosensory 6082 ± 621 Gustatory/Digestive 6422 ± 831 Primary visual 9180 ± 1173 Secondary visual 9256 ± 879 Primary auditory 8595 ± 788 Ventral auditory 7875 ± 438 Dorsal auditory 8249 ± 450 Insular 5636 ± 436 Ectorhinal 7527 ± 403 Entorhinal 7051 ± 548 Perirhinal 6776 ± 674 Total 7081 ± 780
Values are expressed as mean ± SEM. F (24, 50) = 2.15, P = 0.011. N = 3 mice.
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Figure 12. ErbB4-positive Cells In Cortex Are Enriched In Layer 2/3, Are
GABAergic Interneurons and Positive For PV.
(A) Diagram of mouse brain sagittal (top) and coronal (bottom) sections. Coronal section position was indicated by the dashed line in top. ( B) Coronal section of
ErbB4-reporter; GAD67::GFP mouse brain counter-stained with anti-PV, – 1.58 mm relative to bregma. Area shown was indicated in the rectangle in A. ( C)
Coronal section of the primary somatosensory cortex (SS1) spanning layers 1 –
6. Area shown was indicated in the rectangle in B. Arrows, neurons positive for
ErbB4, GAD67 and PV; empty arrows, neurons positive for ErbB4 and GAD67 but not PV; arrowhead, neurons positive for GAD67 and PV but not ErbB4; empty arrowhead, neurons positive for GAD67 alone; asterisks, cells positive for either ErbB4 or PV but not GAD67. CPu, caudate putamen; GI / DI, gustatory and digestive cortex; Hp, hippocampus; Hy, hypothalamus; Ins, insular cortex;
LV, lateral ventricle; M1, primary motor cortex; M2, secondary motor cortex; Pir, piriform cortex; RS, retrosplenial cortex; SS1, primary somatosensory cortex;
SS2, secondary somatosensory cortex; Th, thalamus .
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Table 6. ErbB4-positive Cells In Cortex Are GABAergic Interneurons, Many of Which are PV-positive.
SS1 Layer GAD67 / PV / ErbB4 / PV / ErbB4 / GAD67 / ErbB4 ErbB4 GAD67 GAD67 PV PV
1 100.0 ± 87.7 ± 0.0 0.0 ± 0.0 3.5 0.0 ± 0.0 NA NA 2/3 38.3 ± 62.5 ± 35.6 ± 62.3 ± 91.5 ± 98.7 ± 0.7 1.3 2.4 0.7 3.0 1.3 4 66.6 ± 62.4 ± 53.4 ± 72.9 ± 92.4 ± 97.9 ± 1.3 2.9 5.9 1.5 5.6 2.6 5 55.9 ± 61.9 ± 56.4 ± 52.4 ± 82.2 ± 96.7 ± 1.0 4.3 1.9 5.2 3.5 3.8 6 35.0 ± 65.5 ± 34.3 ± 65.7 ± 95.2 ± 95.6 ± 1.5 6.7 2.9 5.4 2.7 3.0 Total 42.8 ± 64.1 ± 40.6 ± 61.1 ± 88.3 ± 97.9 ± 0.7 0.7 1.8 1.0 2.2 1.6 FrA
1 100.0 ± 79.6 ± 0.0 0.0 ± 0.0 6.4 0.0 ± 0.0 NA NA 2/3 31.7 ± 75.1 ± 24.9 ± 92.2 ± 99.0 ± 99 ± 0.5 9.4 3.6 10.8 4.9 0.7 5 55.9 ± 70.5 ± 52.8 ± 78.3 ± 97.1 ± 98.3 ± 0.3 6.8 4.3 12.2 8.7 1.0 Total 42.2 ± 72.4 ± 39.3 ± 81.1 ± 97.8 ± 98.8 ± 0.2 6.0 2.4 9.3 6.9 0.4
Values are expressed as mean percentage ± SEM. FrA, frontal association cortex; SS1, primary somatosensory cortex. N = 3 mice.
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In both neonatal and adult mice, nearly all ErbB4-positive cells were
GAD67-positive in various layers of both cortices, suggesting ErbB4-positive cells are GABAergic interneurons (Figures 12 and 13 and Tables 6 and 7). In adult mice, ~40% of ErbB4-positive cells were also positive for PV, indicating that they were basket and chandelier subtypes of interneurons (Figure 12C and Table
6). However, only a fraction of GAD67-positive cells were positive for ErbB4. In layer 5, for example, 38.1% of GAD67-positive cells were negative for ErbB4
(Figure 12C and Table 6). These results suggest that a subgroup of GABAergic interneurons in the cortex is a direct target of the NRG1-ErbB4 pathway.
Interestingly, higher co-localization rates were observed in layer 1 (> 80%) for both cortices, suggesting a larger role for NRG1-ErbB4’s regulation in layer 1.
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Table 7. Cells Expressing ErbB4 at P1-7 Are GABAergic Interneurons In
Cortex and Hippocampus.
P1 -7 GAD67 / ErbB4 ErbB4 / GAD67 CX 99.4 ± 0.1 65.9 ± 0.4 CA1 -3 99.2 ± 0.2 60.4 ± 0.3 DG 99.6 ± 0.2 39.2 ± 2.4
Values are expressed as mean percentages ± SEM. CA1-3, CA1-3 areas of hippocampus; Cx, cortex; DG, dentate gyrus of hippocampus; P1-7, postnatal days 1-7.
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Figure 13. ErbB4-positive Cells In Cortex and Hippocampus of Neonatal
Mice are GABAergic Interneurons.
(A) Coronal section of ErbB4-reporter; GAD67::GFP mouse brain. Expression of tdTomato was induced on P1 and examined on P7. ( B) Enlarged area of the squared region in A. Arrows, neurons positive for ErbB4 and GAD67; open arrows, neurons positive for GAD67 alone. CA1, CA1 area of hippocampus; DG, dentate gyrus of hippocampus; P1-7, postnatal days 1-7.
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3.5 Hippocampus
Hippocampus, a sea horse-shaped structure deep to the cortex, is responsible for memory consolidation, retrieval, and spatial navigation (Morris et al., 1982; Zola-Morgan and Squire, 1986). The hippocampus can be divided into
Cornu Ammonis (CA) 1, 2, and 3 areas and the dentate gyrus (DG) (Buzsaki,
2011). Axons and neurons in the hippocampus form a trisynaptic loop. In the perforant pathway, axons from the entorhinal cortex synapse onto granule cells in the granule cell layer of the DG, which relay the information via the mossy fiber pathway to pyramidal neurons in CA3. CA3 pyramidal neurons project to CA1 pyramidal neurons via the Schaffer collateral pathway, which in turn project via subiculum back to the entorhinal cortex (Buzsaki, 2011). ErbB4-positive cells were detectable in various regions of the hippocampus at comparable densities, except the subiculum areas, where ErbB4-positive cells were enriched (Table 8).
In the DG, ErbB4-positive neurons were enriched in the granule cell layer and polymorphic layer, but lower in the molecular layer. ErbB4-positive cell densities varied across the layers of CA1-3 regions. They were localized primarily in stratum pyramidal, where pyramidal neurons are concentrated and stratum lacunosum-moleculare, where perforant path axons synapse onto distal dendrites of pyramidal neurons. The density was low in stratum oriens, where many basket interneurons are found as well as basal dendrites from pyramidal neurons, stratum lucidum, where mossy fibers from DG granule cells pass or stratum radiatum, where Schaffer collaterals are contained (Figure 14B and C).
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Figure 14. ErbB4-positive Cells In Hippocampus Are Concentrated In
Stratum Pyramidal, Are GABAergic Interneurons and Positive For PV.
(A) Diagram of mouse brain sagittal (top) and coronal (bottom) sections. Coronal section position was indicated by the dashed line in top. ( B) Coronal section of
ErbB4-reporter; GAD67::GFP mouse brain counter-stained with anti-PV, – 1.58 mm relative to bregma. Area shown was indicated in the rectangle in A. ( C)
Layers of CA1 and DG areas indicated by rectangle in B. Arrows, neurons positive for ErbB4, GAD67 and PV; empty arrows, neurons positive for ErbB4 and GAD67 but not PV; arrowhead, neurons positive for GAD67 and PV but not
ErbB4; empty arrowhead, neurons positive for GAD67 alone. 3V, third ventricle;
CPu, caudate putamen; Ect, ectorhinal cortex; gr, granular layer; Hp, hippocampus; Hy, hypothalamus; ml, molecular layer; Pir, piriform cortex; pl, polymorph layer; PRh, perirhinal cortex; PtA, parietal association cortex; slu, stratum lucidum; slm, stratum lacunosum-moleculare; so, stratum oriens; sp, stratum pyramidal; stratum radiatum; RS, retrosplenial cortex; RTN, reticular thalamic nucleus; SS1, primary somatosensory cortex; SS2, secondary somatosensory cortex; Th, thalamus.
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Table 8. ErbB4-positive Cell Densities Vary In Hippocampus.
Hippocampal regions ErbB4 cells / mm 3 CA1 3689 ± 129 CA2 5000 ± 25 CA3 3594 ± 630 Dentate gyrus 3279 ± 54 Dorsal subiculum 4918 ± 1108 Ventral subiculum 7907 ± 620 * Presubiculum 7407 ± 1175 Parasubiculum 9329 ± 569 *** Total 4667 ± 338
Values are expressed as mean ± SEM. F (7, 16) = 11.13, P < 0.001. N = 3 mice.
Bonferroni post hoc comparisons vs. total, * P < 0.05, *** P < 0.001.
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Table 9. ErbB4-positive Cells In Hippocampus Are GABAergic
Interneurons, Many of Which Are PV-positive.
CA1 -3 Layer GAD67 PV / ErbB4 / PV / ErbB4 / GAD67 / ErbB4 ErbB4 GAD67 GAD67 PV / PV so 95.6 ± 41.1 ± 52.7 ± 47.9 ± 47.2 ± 98.2 ± 1.7 3.2 0.8 5.6 2.8 1.1 sp 98.9 ± 48.3 ± 84.6 ± 49.6 ± 82.2 ± 98.4 ± 0.7 5.3 1.8 5.8 2.4 1.6 slu 96.9 ± 41.8 ± 71.8 ± 33.7 ± 93.3 ± 100.0 ± 3.1 7.5 8.1 6.8 3.9 0.0 sr 98.7 ± 15.4 ± 56.6 ± 11.2 ± 67.9 ± 82.7 ± 1.3 3.8 2.6 3.2 7.6 11.2 slm 99.6 ± 1.8 ± 87.0 ± 1.5 ± 0.4 1.8 2.5 1.5 NA NA total 98.4 ± 29.8 ± 71.4 ± 29.7 ± 71.0 ± 97.5 ± 0.2 0.7 1.3 1.2 1.42 0.1 DG ml 94.7 ± 4.3 ± 98.2 ± 4.6 ± 100.0 ± 100.0 ± 3.7 1.5 1.8 1.6 0.0 0.0 gr 99.0 ± 16.3 ± 81.5 ± 16.3 ± 86.7 ± 100.0 ± 1.0 2.3 5.5 3.9 8.2 0.0 pl 96.9 ± 5.0 ± 43.0 ± 4.2 ± 66.7 ± 100.0 ± 3.1 5.0 4.5 4.2 0.0 0.0 total 96.9 ± 10.4 ± 75.1 ± 9.7 ± 83.9 ± 100.0 ± 1.2 1.2 1.7 1.2 6.8 0.0
Values are expressed as mean percentage ± SEM. N = 3 mice. DG, dentate gyrus; gr, granule layer; ml, molecular layer; pl, polymorph layer; slm, stratum lacunosum-moleculare; slu, stratum lucidum; so, stratum oriens; sp, stratum pyramidal; sr, stratum radiatum.
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As observed in the cortex, most ErbB4-positive cells in the CA1-3 regions were
GAD67-positive, regardless of layers (Figure 14B and C). 30% of ErbB4-positive cells were positive for PV, whereas 70-80% of PV-positive cells were positive for
ErbB4. In agreement, NRG1 has been shown to suppress LTP via activating
ErbB4 in GABAergic neurons (Huang 2000; Woo 2007; Pitcher 2008; Wen 2010;
Chen 2010).
3.6 Basal Ganglia
Basal ganglia are responsible for modulation of movement and intimately related to reward and motivation. Unique from the rest of the brain, >90% of neurons in the basal ganglia are medium spiny GABAergic neurons. Its nuclei, including the caudate putamen, the nucleus accumbens, and lateral septum receive input from dopaminergic neurons in the substantia nigra and ventral tegmental area in the midbrain. In addition, nuclei of the basal ganglia receive glutamatergic inputs from motor cortices (to caudate putamen), from the sub- thalamic nucleus in the hypothalamus (to globus pallidus), and from the amygdala (to nucleus accumbens) (Mink, 2008; Nolte, 2009f). The motor circuit culminates in inhibitory projections from the internal segment of the globus pallidus to the thalamus where the signal is relayed back to the motor cortices.
ErbB4-positive cells were throughout in the basal ganglia, but enriched in fundus of striatum, bed nucleus of the stria terminalis, diagonal bands nucleus, and ventral tip of the lateral septum (arrow, Figure 15A, B and Table 10), suggesting a role for ErbB4 in the reward circuitry. Expression of ErbB4 was low in ventral
93 pallidum, caudate putamen, and globus pallidus. Most ErbB4-positive cells were
GABAergic although many that were positive for GAD67 were not ErbB4-positive
(Figure 15C and D). This suggests that ErbB4-expressing cells were always
GABAergic in the basal ganglia, but that most GABAergic neurons do not express ErbB4. This disparity was largest in the caudate putamen (Figure 15C).
The sparsity and randomness of ErbB4 expressing cells suggest that these are most likely interneurons that play a modulatory role and not the principal cells of this area. Supporting this notion further, ErbB4-positive neurons did not co- localize with neurogranin (NG) a marker for medium spiny neurons in this area
(Figure 15E and F).
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Figure 15. ErbB4-positive Cells In the Basal Ganglia Show Unique
Distribution and Are GABAergic Interneurons.
(A) Diagram of mouse brain sagittal (top) and coronal (bottom) sections. Coronal section position was indicated by the dashed line in top. ( B) Coronal section of
ErbB4-reporter; GAD67::GFP mouse brain, 0.98 mm relative to bregma. Area shown was indicated in the rectangle in A. ( C) Caudate putamen area indicated by square C’. ( D) Lateral septum area indicated by square D’. Arrows, neurons positive for ErbB4 and GAD67; Empty arrows, neurons positive for GAD67 alone.
(E) Coronal section of ErbB4-reporter; GAD67::GFP mouse brain, 0.98 mm relative to bregma counterstained with neurogranin (NG) in blue. (F) Area indicated by boxed area in E. Arrows, neurons positive for ErbB4 and GAD67;
Empty arrows, neurons positive for GAD67 and NG. Area shown was indicated in the rectangle in A. AcC, nucleus accumbens core; AcSh, nucleus accumbens shell; CPu, Caudate putamen; Cx, Cortex; DB, diagonal band nucleus; LS, lateral septum; LV, lateral ventricle; Tu, olfactory tubercle.
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Table 10. ErbB4-positive Cell Densities Vary In Basal Ganglia.
Basal ganglia regions ErbB4 cells / mm 3 Caudate putamen 2743 ± 407 Nucleus accumbens core 4194 ± 433 Nucleus accumbens shell 5372 ± 618 Claustrum 4562 ± 403 Globus pallidus 707 ± 285 Ventral pallidum 2017 ± 672 Lateral septum 4904 ± 908 Medial septum 2593 ± 903 Diagonal bands nucleus 5582 ± 1808 Substantia innominata 4533 ± 1432 Fundus of striatum 9615 ± 2207 ** Bed nucleus of the stria terminalis 6019 ± 1476 Total 3555 ± 607
Values are expressed as mean ± SEM. F (11, 24) = 4.09, P = 0.002. N = 3 mice.
Bonferroni post hoc comparisons vs. total, ** P < 0.01.
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3.7 Amygdala
The amygdala is implicated in modulation of fear and emotional memory.
It has two input pathways. First, information from sensory cortices goes to the basal lateral amygdala, which relays the signal to the central amygdala via the intercalated nucleus (Figure 16A and B). Neurons in the central amygdala project to monoaminergic cells of the midbrain and hindbrain and to cells in hypothalamus. Second, the signal from sensory cortices passes from the lateral amygdala to the ventral portion of the basal lateral amygdala or basal amygdala.
From here, the signal is relayed to the nucleus accumbens. The effect is to modulate arousal and emotional state and encode them into memory associated with certain stimuli (LeDoux, 2008). Among all brain areas, amygdala was the region that had the highest ErbB4-positive cell density (Table 2), in agreement with in situ hybridization analysis (Lai 1992; Woo 2007). In amygdala, ErbB4- positive cell density varied significantly in different sub-regions with the highest concentration in the intercalated nucleus (Table 11, Figure 16B and D). Almost all ErbB4-positive cells in amygdala were GAD67-positive (Figure 16), except a few cells in the medial amygdala (Figure 16E). Some GAD67-positive cells were not positive for ErbB4, as observed in other brain regions (Figure 16C-E). These results suggest that ErbB4 expressing cells in the amygdala are mostly
GABAergic interneurons although not all GABAergic interneurons express ErbB4 and a small population of ErbB4 expressing cells, in particular those in the medial amygdala, may be non-GABAergic.
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Figure 16. ErbB4-positive Cells In the Amygdala Are Concentrated In the
Intercalated Nucleus.
(A) Diagram of mouse coronal section. The position was indicated by the dashed line in Figure 12A. ( B) Amygdala of ErbB4-reporter; GAD67::GFP mouse brain, –
1.58 mm relative to bregma. Dashed lines demarcate nuclei of the amygdala.
Areas in squares C’, D’ and E’ were shown in panels C, D and E. Arrows, neurons positive for ErbB4 and GAD67; Empty arrows, neurons positive for
GAD67 alone; Arrowheads, neurons positive for ErbB4 alone. BLA, basal lateral amygdala; BMA, basal medial amygdala; CeA, central amygdala; Hp, hippocampus; Hy, hypothalamus; IA, intercalated nucleus of the amygdala; MA, medial amygdala; SS1, primary somatosensory cortex; Th, thalamus.
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Table 11. ErbB4-positive Cells Are Enriched In the Intercalated Nucleus and
Medial Amygdala, Within the Amygdala.
Amygdala regions ErbB4 cells / mm 3 Basal lateral amygdala 8609 ± 1058 Basomedial amygdala 8254 ± 1675 Medial amygdala 13600 ± 2120 Central amygdala 2834 ± 552 Amygdala cortex 10190 ± 694 Anterior amygdala area 6874 ± 1665 Posterior amygdala area 13590 ± 1626 Intercalated nucleus 30800 ± 5365 *** Total 10150 ± 795
Values are expressed as mean ± SEM. F (7, 16) = 13.10, P < 0.001. N = 3 mice.
Bonferroni post hoc comparisons vs. total, *** P < 0.001.
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3.8 Thalamus
The dorsal thalamus acts as a relay center for sensory information traveling to and from sensory cortices other than olfaction (Figure 17A and B).
Information from the cerebellum, basal ganglia and limbic structures is also relayed at the ventral thalamus. ErbB4-positive cell density was low in the thalamus (Figure 10E, 17B and Table 12). They are mainly distributed in the medial habenula, in agreement with ErbB4 mRNA localization (Lai and Lemke,
1991; Steiner et al., 1999; Bruce et al., 2002; Woo et al., 2007). Only a small fraction of ErbB4-positive cells were GAD67-positive; more appeared to be negative for GAD67. However, almost all GAD67-positive cells in the medial habenula are ErbB4-positive (Figure 17D). I observed ErbB4-positive cells that are scattered in the central thalamus. Their soma size was small, but had radiating processes that look more like glial cells (Figure 17B and C asterisks).
These cells were positive for S100β but not for GFAP, Olig2, or MBP (Figure 18).
The identity and function of these cells remains unclear. They may be GFAP- negative astrocytes or unidentified oligodendrocytes. They appeared prominent in the thalamus because of low background ErbB4 signal. Careful examination indicated that they were detectable in the hypothalamus, midbrain, and hindbrain
(asterisks in Figure 17, and figures hereafter). The majority of GABAergic neurons in the thalamus are concentrated in the reticular thalamic nucleus that projects to and inhibits glutamatergic neurons within the thalamus (Hendry, 2008;
Nolte, 2009e). In agreement, most GAD67-GFP cells were strictly localized in the reticular thalamic nucleus. Previous studies indicated that ErbB4 mRNA is
103 enriched in the nucleus (Woo 2007; Lai 1992). Intriguingly, most GAD67-positive cells were not positive for ErbB4 in the region (Figure 17C).
Figure 17. ErbB4-positive Cells Are Enriched In Medial Habenula.
(A) Diagram of mouse brain sagittal (top) and coronal (bottom) sections. Coronal section position was indicated by dashed line. ( B) Coronal section of ErbB4- reporter; GAD67::GFP mouse brain, – 1.58 mm relative to bregma. Position of the section was indicated by rectangle in A. Asterisks mark ErbB4-positive cells with non-neuronal morphology. ( C) Reticular thalamic nucleus area indicated by square in B. ( D) Coronal section of medial habenula, – 1.58 mm relative to bregma. Position of the section is indicated by smaller box in A. Arrows, neurons positive for ErbB4 and GAD67; Empty arrows, neurons positive for GAD67 alone;
Arrowheads, neurons positive for ErbB4 alone. CPu, caudate putamen; Ect, ectorhinal cortex; Hp, hippocampus; Hy, hypothalamus; LH, lateral habenula;
MH, medial habenula; Th, thalamus. Pir, piriform cortex; PRh, perirhinal cortex;
PtA, parietal association cortex; SS1, primary somatosensory cortex; SS2, secondary somatosensory cortex; RS, retrosplenial cortex; RTN, reticular thalamic nucleus.
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Table 12. ErbB4-positive Cells Are Enriched In the Medial Habenula Of the Thalamus.
Thalamus regions ErbB4 cells / mm 3 Paraventricular thalamic nucleus 2860 ± 936 Central medial thalamic nucleus 342 ± 150 Reuniens thalamic nucleus 1798 ± 470 Reticular thalamic nucleus 1267 ± 429 Rhomboid thalamic nucleus 535 ± 70 Anterior thalamic area 414 ± 155 Posterior thalamic nucleus 949 ± 603 Submedius 869 ± 482 Stria medullaris 5451 ± 4024 Mediodorsal thalamic nucleus 573 ± 231 Laterodorsal thalamic nucleus 834 ± 377 Ventromedial thalamic nucleus 415 ± 54 Ventrolateral thalamic nucleus 276 ± 171 Ventroposterior thalamic nucleus 403 ± 51 Medial geniculate 952 ± 407 Lateral geniculate 2598 ± 814 Posterior intralaminar thalamic nucleus 2664 ± 1283 Medial habenula 8832 ± 2655 *** Lateral habenula 2538 ± 862 Intermediodorsal nucleus 629 ± 317 Parafascicular nucleus 2277 ± 857 Total 1245 ± 397
Values are expressed as mean ± SEM. F (20, 42) = 3.01, P = 0.001; F (19, 40) = 1.51,
P = 0.134 excluding medial habenula. N = 3 mice. Bonferroni post hoc comparisons vs. total, *** P < 0.001.
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Figure 18. S100β Was Detected In ErbB4-positive Cells In Thalamus,
Hypothalamus, Midbrain and Hindbrain.
(A, B) ErbB4-positive cells in thalamus, with small soma and radiating processes.
(a, b) Enlarged images of ErbB4-postive cells in rectangles in A and B respectively. These cells were detected in low density in areas including hypothalamus (Figure 19), midbrain (Figure 22) and hindbrain (Figure 24) as well as thalamus (Figure 10E, 17B). Arrows, cells positive for ErbB4 and S100β;
Empty arrows, cells positive for S100β and GFAP but negative for ErbB4;
Arrowheads, cells positive for ErbB4 alone. GFAP, glial fibrillary acidic protein;
MBP, myelin basic protein; Olig2, Oligodendrocyte transcription factor two;
S100β, s100 calcium binding protein beta.
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3.9 Hypothalamus
The hypothalamus receives input from nearly all sensory modalities and from hippocampus, amygdala, and cingulate cortex. It also contains sensors for blood temperature, sugar, mineral, and hormone levels (Nolte, 2009d). It plays integral roles in regulating metabolism, thermoregulation, sleep, stress response, and sexual behavior. ErbB4-positive cell densities varied in the hypothalamus, being especially high in the paraventricular hypothalamic nucleus (PaVH), dorsal medial hypothalamic nucleus (DM), and posterior hypothalamic nucleus (Figure
10E, 19A, B, C and Table 13). The expression profile generally agrees with that of previous in-situ reports (Lai and Lemke; Ma et al., 1999; Woo et al.). As observed in other areas, some, but not all, ErbB4-positive cells in the PaVH and
DM were positive for GAD67 (Figure 19D) (data not shown). Likewise, some
GAD67-positive cells did not express ErbB4. These results suggest that ErbB4- positive cells in the hypothalamus were a mixture of GABAergic interneurons and non-GABAergic cells (Figure 19D). Neurons in the PaVH are known to release oxytocin, a factor implicated in social bonding, sexual response, maternal care, and lactation (Lee et al., 2009). Co-staining analysis indicated that ~50% of oxytocin-expressing cells in the hypothalamus were positive for ErbB4, suggesting that NRG1-ErbB4 signaling may regulate sociality or maternal instincts (Figure 20A, B and D).
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Table 13. ErbB4-positive Cells Are Enriched In PaVH.
Hypothalamus regions ErbB4 cells / mm 3 Medial preoptic nucleus 5200 ± 595 Lateral preoptic nucleus 4746 ± 1483 Median preoptic nucleus 4067 ± 586 Ventromedial hypothalamic nucleus 3308 ± 788 Dorsomedial hypothalamic nucleus 13640 ± 1441 Posterior hypothalamic nucleus 13780 ± 3773 Anterior hypothalamic nucleus 6614 ± 1969 Lateral hypothalamic nucleus 3825 ± 1388 Premammillary nucleus 8008 ± 1671 Medial mammillary nucleus 703 ± 189 Supramammillary nucleus 8165 ± 2965 Arcuate nucleus 7824 ± 1744 Periventricular nucleus 8645 ± 1431 Suprachiasmatic nucleus 7616 ± 2183 Paraventricular hypothalamic nucleus 15820 ± 4529 ** Tuber cinereum area 2163 ± 369 Zona incerta 1819 ± 473 Median eminence 7323 ± 1855 Subthalamic nucleus 8865 ± 1877 Total 5306 ± 1178
F(18, 38) = 4.34, P < 0.001. N = 3 mice. Post hoc comparisons vs. total, ** P < 0.01.
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Figure 19. ErbB4-positive Cells Enriched In the Paraventricular and
Dorsomedial Hypothalamic Nuclei.
(A) Diagram of mouse brain sagittal (top) and coronal (middle and bottom) sections. Coronal section positions were indicated by dashed lines b and c. ( B)
Coronal section of ErbB4-reporter; GAD67::GFP mouse brain, - 1.06 mm relative to bregma. Area shown was indicated by the rectangle in A middle.
Hypothalamus nuclei were demarcated by dashed lines. ( C) Coronal section of
ErbB4-reporter; GAD67::GFP mouse brain, - 1.58 mm relative to bregma.
Asterisks mark ErbB4-positive cells with non-neuronal morphology.
Hypothalamus nuclei were demarcated by dashed lines. ( D) Paraventricular hypothalamic nucleus (PaVH), enlarged from the area in the square. Arrows, neurons positive for ErbB4 and GAD67; Empty arrows, neurons positive for
GAD67 alone; Arrowheads, neurons positive for ErbB4 alone. AH, anterior hypothalamic nucleus; Arc, arcuate nucleus; CPu, caudate putamen; Cx, cortex;
DM, dorsomedial hypothalamic nucleus; Hp, hippocampus; Hy, hypothalamus;
LH, lateral hypothalamus; MA, medial amygdala; PaVH, paraventricular hypothalamic nucleus; RTN, reticular thalamic nucleus; Th, thalamus; VM, ventral medial hypothalamic nucleus; ZI, zona incerta.
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Figure 20. ErbB4-positive Cells Co-localize With Oxytocin But Not
Vasopressin In the Paraventricular Hypothalamic Nucleus.
(A) Diagram of mouse brain sagittal (top) and coronal (bottom) sections. Coronal section position was indicated by the dashed line in top. ( B, C) Coronal section of
ErbB4-reporter mouse brain, – 1.06 mm relative to bregma. Shown was the
PaVH area, indicated by the rectangle in A. Sections were stained with antibodies against oxytocin (B) or vasopressin (C). ( D, E) PaVH areas stained with anti-oxytocin (D) or anti-vasopressin (E). Areas shown were indicated by squares in B and C, respectively. Arrows, neurons positive for ErbB4 and oxytocin; Empty arrows, neurons positive for oxytocin or vasopressin alone;
Arrowheads, neurons positive for ErbB4 alone. 3V, third ventricle; CPu, caudate putamen; Cx, cortex; Hp, hippocampus; Hy, hypothalamus; PaVH, paraventricular hypothalamic nucleus; RTN, reticular thalamic nucleus; Th, thalamus.
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In contrast, none of the cells expressing vasopressin, a factor related to oxytocin sometimes opposing its effects, were positive for ErbB4 (Figure 20C and E)
(Keverne and Curley, 2004). Together, these results suggest that NRG1-ErbB4 signaling may regulate metabolism or other hypothalamus-associated functions.
3.10 Midbrain
The midbrain is located below and behind the cerebral cortex and in front of the hindbrain. In the midbrain, ErbB4-positive cells are clustered in superior colliculus, periaqueductal gray, raphe nuclei, and interpeduncular nucleus
(Figures 21-23, and Table 14). Overall, most GAD67-positive cells were negative for ErbB4 in midbrain (Figure 21A and B). Among the cells that were positive for
ErbB4, some were GAD67-positive but many were not (Figure 21C).
Interestingly, ErbB4-positive cells were localized in regions that are enriched in monoaminergic neurons, including substantia nigra and ventral tegmental area (VTA), where dopamine neurons are concentrated (Figures 21B,
D and 22). This is consistent with previous in-situ reports that dopamine neurons express ErbB4 (Steiner et al., 1999; Abe et al., 2009; Zheng et al., 2009). The dopaminergic neurons in the substantia nigra pars compacta (SNc) project to the caudate putamen in the basal ganglia and are implicated in motor control. On the other hand, dopamine neurons in the ventral tegmental area (VTA) project to nucleus accumbens for reward and motivation. They also project to cortical and limbic structures for cognition. The serotoninergic neurons in raphe nuclei have been implicated in alertness and mood regulation (Nolte, 2009c). In both VTA
115 and SNc, ~50% of ErbB4-positive cells were positive for tyrosine hydroxylase, a marker of dopaminergic neurons (Nagatsu, 1995) (Table 15 and figure 22). Less than 10% of ErbB4-positive cells were GAD67-positive in both regions. However,
ErbB4-positive cells accounted for only a fraction (7-22%) of dopaminergic neurons (Table 15). This is inconsistent with the notion that nearly all dopamine neurons express ErbB4 (Abe et al., 2009; Zheng et al., 2009). These data suggest that ErbB4 expressing cells in the VTA and SNc are often dopaminergic and sometimes GABAergic.
Having found high densities of ErbB4-positive cells in many raphe nuclei, I examined if they were serotoninergic (5-HT) neurons by staining with anti-5-HT antibody. I focused on the rostral linear and dorsal raphe nuclei of the midbrain and the median raphe nucleus of the hindbrain, where large amounts of 5-HT neurons are located. ErbB4-positive cells were indeed positive for 5-HT, indicating that ErbB4 is expressed in serotoninergic neurons (Figure 23).
Quantitatively, 20-40% of ErbB4-positive cells were positive for 5-HT.
Conversely, 10-30% of 5-HT cells expressed ErbB4 (Table 15). In the raphe nuclei, most GAD67-positive cells were not positive for ErbB4. These data indicate that ErbB4 is expressed in serotoninergic neurons in raphe nuclei, identifying a novel cellular target of NRG1-ErbB4 signaling.
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C’
D’
Figure 21. ErbB4-positive Cells In the Midbrain.
(A) Diagram of mouse brain sagittal (top) and coronal (bottom) sections. Coronal section position was indicated by the dashed line in top. ( B) Coronal section of
ErbB4-reporter; GAD67::GFP mouse brain, – 3.08 mm relative to bregma.
Midbrain nuclei were demarcated by dashed lines. ( C, D) Superior colliculus and ventral tegmental area, indicated by square C’ and D’ in B, respectively. Arrows, neurons positive for ErbB4 and GAD67; Empty arrows, neurons positive for
GAD67 alone; Arrowheads, neurons positive for ErbB4 alone. Cx, Cortex; Hp, hippocampus; IF, interfascicular nucleus; MM, medial mammillary nucleus; PAG, periaqueductal grey; SC, superior colliculus; SNc, substantia nigra compact part;
SNr substantia nigra reticular part; VTA, ventral tegmental area.
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Table 14. ErbB4-positive Cells Are Enriched In Raphe Nuclei In the
Midbrain.
Midbrain regions ErbB4 cells / mm 3 Ventral tegmental area 3459 ± 1115 Substantia nigra pars compacta 2457 ± 694 Substantia nigra pars reticulata 1263 ± 354 Interpeduncular nucleus 9893 ± 3456 * Interfascicular raphe nucleus 14430 ± 4055 *** Rostral linear raphe nucleus 11700 ± 3145 ** Central linear raphe nucleus 3977 ± 2945 Dorsal raphe nucleus 10500 ± 2167 * Red nucleus 1146 ± 307 Periaqueductal gray 4412 ± 1535 Pretectal area 2611 ± 610 Zonal layer of superior colliculus 3408 ± 1066 Superficial gray layer of superior colliculus 2093 ± 644 Optic nerve layer of superior colliculus 3255 ± 812 Intermediate gray layer of superior colliculus 3609 ± 1349 Intermediate white layer of superior colliculus 1590 ± 604 Deep gray layer of superior colliculus 2547 ± 932 External cortex of the inferior colliculus 645 ± 255 Brachium of the inferior colliculus 1438 ± 406 Central nucleus of the inferior colliculus 465 ± 244 Dorsal cortex of the inferior colliculus 1227 ± 471 Midbrain reticular nucleus 939 ± 307 Total 2271 ± 634
Values are expressed as mean ± SEM. F (21, 44) = 5.48, P < 0.001. N = 3 mice.
Bonferroni post hoc comparisons vs. total, * P < 0.05, ** P < 0.01, *** P < 0.001.
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Figure 22. ErbB4-positive Cells Co-localize With Tyrosine Hydroxylase In
Midbrain.
(A) Diagram of mouse brain sagittal (top) and coronal (bottom) sections. Coronal section position was indicated by the dashed line in top. ( B) Coronal section of
ErbB4-reporter; GAD67::GFP mouse brain, – 3.80 mm relative to bregma.
Asterisks marked non-neuronal ErbB4-positive cells. (C, D) Ventral tegmental area (VTA) and substantia nigra compact part (SNc), indicated by squares C’ and
D’, respectively. Arrows, neurons positive for ErbB4 and TH; Empty arrows, neurons positive for TH alone; Arrowheads, neurons positive for ErbB4 alone.
Cx, Cortex; Hp, hippocampus; IPN, interpeduncular nucleus; PAG, periaqueductal grey; PN, pontine nucleus; RLi, rostral linear nucleus; RN, red nucleus; SC, superior colliculus; SNc, substantia nigra compact part; SNr substantia nigra reticular part; VTA, ventral tegmental area.
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Table 15. ErbB4-positive Cells Co-localize With Monoamines.
5-HT / TH / GAD67 / ErbB4 / ErbB4 / ErbB4 / ErbB4 ErbB4 ErbB4 5-HT TH GAD67 VTA 53.7 ± 3.5 10.3 ± 4.2 22.4 ± 8.2 15.0 ± 6.4 SN 44.9 ± 2.2 2.4 ± 2.4 6.9 ± 2.8 3.0 ± 3.0 RLi 29.7 ± 0.0 ± 0.0 30.8 ± 0.0 ± 0.0 14.9 11.1 MnR 18.8 ± 7.1 10.6 ± 5.3 9.4 ± 5.1 5.2 ± 2.7 DR 42.2 ± 3.0 16.4 ± 5.3 20.4 ± 5.0 22.2 ± 4.0 LC 0.0 ± 0.0 45.8 ± 0.0 ± 0.0 6.9 ± 2.2 20.8
Values are expressed as mean percentages ± SEM. N = 3 mice. 5-HT, serotonin;
DR, dorsal raphe; LC, locus coeruleus; MnR, median raphe; RLi, rostral linear raphe nucleus; SN, substantia nigra; TH, tyrosine hydroxylase; VTA, ventral tegmental area.
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Figure 23. ErbB4-positive Cells Co-localize With Serotonin Neurons In
Raphe Nuclei.
(A) Diagram of mouse brain sagittal (top) and coronal (bottom) sections. Coronal section position was indicated by the dashed line in top. ( B) Coronal section of
ErbB4-reporter mouse brain, – 4.36 mm relative to bregma. Sections were stained with anti-5-HT antibody (green). ( C, D) Dorsal raphe of the midbrain and median raphe of the hindbrain, indicated by squares C’ and D’, respectively.
Arrows, neurons positive for ErbB4 and 5-HT; Empty arrows, neurons positive for
5-HT alone; Arrowheads, neurons positive for ErbB4 alone. DR, dorsal raphe; LL, lateral lemniscus; MnR, median raphe; PAG, periaqueductal grey; PN, pontine nucleus; SC, superior colliculus.
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3.11 Hindbrain and Cerebellum
The density of ErbB4-positive cells was low in the hindbrain, compared with other brain areas (Table 2). In hindbrain, ErbB4-positive cells were concentrated in the median raphe (Figure 23), parabrachial nucleus, locus coeruleus (Figure 24), supratrigeminal nucleus, and posterodorsal tegmental area (Table 16).
Most GAD67-positive cells were negative for ErbB4 in hindbrain (Figure
25A-C). Among the cells that were positive for ErbB4, ~45% were GAD67- positive, for example, in locus coeruleus (Table 15). Although ErbB4-positive cells in the midbrain could be monoaminergic, those in the hindbrain were negative for TH, that marks norepinephrine neurons (Nagatsu, 1995) (Figure 24 and Table 15). These results suggest that ErbB4 expression in monoaminergic neurons is neurotransmitter specific.
Previous studies utilizing in-situ hybridization suggested that ErbB4 is expressed in the cerebellum (Ozaki et al., 1997; Rio et al., 1997; Ozaki et al.,
1998; Rieff et al., 1999). Compared with other brain areas, ErbB4-positive cells were fewer in the cerebellum (Table 2 and 17). As shown in Figure 25, GAD67 expression marked Purkinje cells and some smaller interneurons in the granule layer. Occasionally, ErbB4-positive cells were observed in the granule layer.
GAD67 signal was not observed in these ErbB4-positive cells (Figure 25D).
These data suggest that GABAergic neurons of the cerebellum may not express
ErbB4.
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Figure 24. ErbB4-positive Cells Do Not Co-localize With Tyrosine
Hydroxylase In the Locus Coeruleus.
(A) Diagram of mouse brain sagittal (left) and coronal (right) sections. Coronal section position was indicated by the dashed line at left. ( B) Coronal section of
ErbB4-reporter mouse brain, – 5.34 mm relative to bregma. Sections were stained with anti-TH antibody (green). Asterisks marked ErbB4-positive non- neuronal cells. ( C) Locus coeruleus (LC), indicated by the square in B. Arrows, neurons positive for TH alone; Arrowheads, neurons positive for ErbB4 alone.
4V, fourth ventricle; IC, Inferior colliculus; LC, locus coeruleus; Pb, parabrachial nucleus; PCG, pontine central grey.
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Table 16. ErbB4-positive Cell Density Varies In Hindbrain Despite Low
Levels.
Hindbrain regions ErbB4 cells / mm 3 Pontine central gray 1077 ± 140 Reticulotegmental nucleus of pons 1148 ± 444 Pontine reticular nucleus 731 ± 176 Median raphe nucleus 2987 ± 918 Paramedian raphe nucleus 1585 ± 515 Periolivary region 335 ± 113 Nucleus of raphe magnus 1301 ± 290 Principal sensory trigeminal nucleus 5n 2125 ± 652 Motor trigeminal nucleus 5n 769 ± 261 Supratrigeminal nucleus 5n 3882 ± 1974 * Spinal trigeminal nucleus 5n 635 ± 85 Parvocellular reticular nucleus 1414 ± 838 Intermediate reticular nucleus 513 ± 115 Dorsomedial tegmental area 758 ± 162 Posterodorsal tegmental area 2995 ± 848 Medial vestibular 8n 808 ± 231 Lateral vestibular 8n 1147 ± 393 Superior vestibular 8n 691 ± 322 Facial nucleus 7n 492 ± 118 Gigantocellular reticular nucleus 468 ± 71 Medial longitudinal fasciculus 1010 ± 327 Lateral lemniscus 500 ± 217 Parabrachial nucleus 2992 ± 497 Abducens nucleus 6n 319 ± 142 Cochlear 8n 830 ± 182 Locus Coeruleus 2700 ± 456 Nucleus Prepositus 1333 ± 501 Total 1034 ± 270
Values are expressed as mean ± SEM. F (26, 54) = 3.02, P < 0.001. N = 3 mice.
Bonferroni post hoc comparisons vs. total, * P < 0.05.
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Figure 25. ErbB4-positive Cells In the Cerebellum and Hindbrain.
(A) Diagram of mouse brain sagittal (top) and coronal (bottom) sections. Coronal section position was indicated by the dashed line in top. ( B) Coronal section of
ErbB4-reporter; GAD67::GFP mouse brain, – 5.52 mm relative to bregma. ( C, D)
Spinal trigeminal nucleus and simple lobule of cerebellum. Areas shown were indicated by squares C’ and D’ in B. Arrows, neurons positive for ErbB4 and
GAD67; Empty arrows, neurons positive for GAD67 alone; Arrowheads, cells positive for ErbB4 alone. 4V, fourth ventricle; 7N, facial nucleus; Cch, cochlear nucleus; Cul, culmen lobule; Crus 1; Crus 1 of the ansiform lobule; GI, gigantocellular reticular nucleus; IC, inferior colliculus; Ling, lingual lobule; PFI, paraflocculus lobule; RMg, raphe magnus nucleus; Sim, simple lobule; SP5, spinal trigeminal nucleus; Ve, vestibular nucleus.
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Table 17. Slight Variation of ErbB4-positive Cell Densities In Cerebellum.
Cerebellum regions ErbB4 cells / mm 3 Lingula (II) lobule 3744 ± 1304 Central (III) lobule 3460 ± 1838 Culmen (IV / V) lobule 2219 ± 645 Central simple (VI) lobule 1375 ± 259 Lateral simple (VI) lobule 1033 ± 555 Crus 1 of the ansiform (VII) lobule 933 ± 245 Crus 2 of the ansiform (VII) lobule 595 ± 269 Paraflocculus (VIII / IX) lobule 719 ± 158 Central flocculus (X) lobule 1091 ± 399 Lateral flocculus (X) lobule 547 ± 45 Fastigial nucleus 708 ± 204 Interposed nucleus 461 ± 293 Dentate nucleus 1010 ± 495 Total 1497 ± 469
Values are expressed as mean ± SEM. F (12, 26) = 2.35, P = 0.034. N = 3 mice.
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3.12 White Matter Areas and Choroid Plexus
ErbB4-positive cells were observed in the white matter including the corpus callosum (Figure 26A and B). The positive cell density did not vary across white matter areas (Table 18). Interestingly, most ErbB4-positive cells in the corpus callosum were GAD67-positive. However, there were also cells that were positive for GAD67, but not ErbB4 (Figure 26B). Finally, large ErbB4-positive cells were observed in the choroid plexus, a tissue that generates the cerebrospinal fluid (CSF). These mostly co-localized with GAD67 (Figure 26C).
This result suggests that NRG1-ErbB4 signaling may play a role in CSF production although this is not clear.
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Figure 26. ErbB4-positive Cells In the Corpus Callosum and Choroid
Plexus.
(A) Diagram of mouse brain coronal sections, – 0.94 mm relative to bregma.
Coronal section position was indicated by the dashed line in Figure 20A. ( B, C)
Corpus callosum (cc) and choroid plexus in the lateral ventricle. Areas shown were indicated by squares B’ and C’, respectively. Arrows, cells positive for
ErbB4 and GAD67; Empty arrows, cells positive for GAD67 alone.
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Table 18. ErbB4-positive Cells Are Evenly Distributed In White Matter Areas
White matter areas ErbB4 cells / mm 3 Corpus callosum 864 ± 181 Anterior commissure 1373 ± 152 Internal Capsule 1001 ± 285 External capsule 981 ± 201 Fimbria 1142 ± 295 Medial lemniscus 415 ± 184 Cerebral peduncle 969 ± 470 Cerebellar peduncle 486 ± 177 Arbor vitae 533 ± 111 Cerebellar commissure 672 ± 88 Total 925 ± 199
Values are expressed as mean ± SEM. F (9,20) = 1.69, P = 0.432. N = 3 mice.
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3.13 Neuregulin-1 Enhances Dopamine and Serotonin Release.
Having confirmed the presence of ErbB4 in dopamine neurons of the VTA and SNc, and having found the novel cellular target serotonin neurons of the raphe nuclei, I next wanted to ask what function NRG1-ErbB4 signaling serves in these locations. Previous studies had demonstrated that NRG1 treatment could enhance neurotransmitter release in GABAergic synaptosomes when treated in conjunction with high potassium (Woo et al., 2007), in dopaminergic fibers in the hippocampus (Kwon et al., 2008), and in dopaminergic fibers in the striatum when cells in the SNc where treated (Yurek et al., 2004). The medial prefrontal cortex (PFC) is an area that is innervated by norepinephrine, dopamine, and serotonin fibers (Figure 1) (Nolte, 2009c), making it a good test bed for the hypothesis that NRG1 perfusion to this area can enhance the release of dopamine and serotonin, but not norepinephrine.
To test this idea, mice were first cannulated in medial PFC (Prelimbic and
Infralimbic cortices) before having microdialysis probes inserted. The microdialysis probes allow for artificial cerebral fluid (ACSF) and ACSF + NRG1 to be perfused in and for extracellular fluid to defuse across the membrane to be collected as samples (Figure 27). The samples were then analyzed using high performance liquid chromatography (HPLC) using a cation affinity column to separate monoamines. To test the feasibility of this method a pilot study was first conducted using Imipramine, a tricyclic antidepressant, to enhance the levels of norepinephrine, dopamine, and serotonin in the extracellular fluid.
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Figure 27. Experimental Design
(A) Diagram of microdialysis probe depicting ACSF and NRG1 flowing out and dopamine (DA), norepinephrine (NE), and serotonin (5-HT) from extracellular fluid flowing in. ( B) Timeline for experiment. ACSF was perfused in for one hour,
ACSF + NRG1 for 20 min., followed by 3 hours of ACSF. Samples were taken every 20 min. ( C) Diagram of a coronal brain section with red rectangle depicting position of microdialysis probe placement. ( D) Diagram of a sagittal brain section with red rectangle depicting position of microdialysis probe placement (Paxinos and Franklin, 2001). ( E) Image of mouse brain post experiment with hole in medial PFC where probe was located. ( F) Serial sections of mouse brain post experiment. ( G) Dark field image of mouse brain section post experiment with hole in tissue demonstrating where the probe was located.
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Imipramine (10 mg/kg I.P.) was able to drastically enhance levels of all three monoamines after a delay of about 40 min. (Figure 28A).
Next, NRG1 (3.33 µM x 20 min ~ 0.5 µg) was perfused into the medial
PFC of mice, and response to monoamine release was observed. NRG1 was unable to modify norepinephrine release (Figure 28B) with time not affecting
2 levels F (12, 15) = 0.46, P = 0.935, and no linear trend observed post hoc. R =
0.001, P = 0.624. However, NRG1 perfusion was able to increase the release of dopamine beginning at 20 min. post perfusion and continuing through 120 min. post perfusion (Figure 28C and E). There was a statistically significant change in dopamine release over time F (12, 13) = 2.13, P = 0.018 and a highly significant linear trend R 2 = 0.038, P < 0.001. Serotonin release was also enhanced after
NRG1 perfusion. However, the increase was delayed, not beginning until 60 min. post infusion and coming back to near baseline levels at 160 min. post infusion
(Figure 28D and F). The difference across time failed to meet statistical significance by repeated measures ANOVA F (12, 15) = 1.585, P = 0.099. However, the post hoc analysis for linear trend did meet statistical significance R 2 = 0.017,
P = 0.018. These results provide modest evidence that NRG1 can enhance the release of dopamine and serotonin in the medial PFC.
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Figure 28. NRG1 Induces Dopamine and Serotonin Release.
(A) Chromatograph with a baseline reading in black and response to Imipramine
(10 mg/kg) 40-60 min. post injection in red. Inset zoomed chromatograph to highlight baseline levels. (B) Quantified data demonstrating NRG1 perfusion was unable to modify norepinephrine release. ( C) Quantified data demonstrating
NRG1 perfusion increased dopamine release. ( D) Quantified data demonstrating
NRG1 perfusion increased 5-HT release after a delay. ( E and F)
Chromatographs with a baseline reading in red and response to NRG1 perfusion
40 and 80 min. post perfusion respectively in black.
4. DISCUSSION
Through the use of ErbB4-reporter mice, where tdTomato red fluorescent protein was expressed in cells expressing ErbB4, I was able to demonstrate that ErbB4 is widely expressed throughout the adult brain. However, there were obvious regional differences. Notably, ErbB4-positive cell density was highest in amygdala, followed by cortex, and hypothalamus. ErbB4-positive cell density was conspicuously low in other regions such as thalamus, hindbrain and cerebellum.
By crossing the ErbB4-reporter mice with mice that express a green fluorescent protein in GABAergic cells expressing GAD67, I was able to determine that nearly all (≥ 98% in cortex and hippocampus) ErbB4-positive cells in cortex, hippocampus, basal ganglia and most of amygdala were GABAergic. Similar results were obtained when I induced tdTomato expression neonatally.
Surprisingly, the hybrid mice revealed that ErbB4-positive cells in subcortical regions were not always GABAergic. Using immunofluorescent staining in conjunction with the ErbB4-reporter mice I was able to determine that
ErbB4 was expressed in oxytocin neurons in the paraventricular hypothalamic nucleus. Cells with non-neuronal morphology were present throughout subcortical areas but most evident in the thalamus. These cells were positive for
S100β, but not other glial markers. ErbB4-positive cells were present in monoaminergic nuclei. These cells were sometimes positive for tyrosine
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140 hydroxylase in the ventral tegmental area (VTA) and substantia nigra compacta
(SNc) indicating they are dopamine neurons, but not in the locus coeruleus suggesting ErbB4 is not expressed in norepinephrine neurons. ErbB4-positive cells were particularly enriched in several Raphe nuclei and sometimes were immunoreactive to antibodies against serotonin. Finally, by perfusing Neuregulin-
1 (NRG1) into the medial prefrontal cortex (PFC) I was able to provide modest evidence that NRG1 can enhance the release of dopamine and serotonin but not norepinephrine.
4.1 ErbB4-positive Cells In the Olfactory Bulb And Deficits In Olfaction
Seen In Schizophrenia
ErbB4-positive cells were observed in the olfactory bulb including in the mitral cell layer were the principal cells are located. ErbB4-positive cells were also located in olfactory associated areas like the accessory olfactory bulb and the piriform cortex. These observations are of particular interest as it has been noted that schizophrenics are sometimes impaired in the perception of smell, having difficulty differentiating different odors (Kastner et al., 2013). Additionally, abnormalities have been observed in the olfactory epithelium tissue itself
(Pantazopoulos et al., 2013). It is possible that polymorphisms on either NRG1 or
ErbB4 could either alter the function or the expression level of these genes. This then could lead to dysfunction in either the ErbB4-positive olfactory neurons or
ErbB4-positive cells in the olfactory associated areas that are involved in the
141 perception of odors. Thus altered NRG1-ErbB4 signaling may be directly involved in olfactory deficits observed in schizophrenia.
4.2 ErbB4-positive Cells Are Enriched In the Intercalated Nucleus Of the
Amygdala and Blunted Affect Seen In Schizophrenia
ErbB4-positive cell density was highest in the amygdala. However, there was significant variation within the amygdala, with the intercalated nucleus having a density several fold higher than any other subregion in the brain. The intercalated nucleus plays a unique role in the amygdala gating information passing from the lateral and basal amygdala to the central amygdala (LeDoux,
2008). As sensory information flows into the lateral amygdala it passes to the basal and to the central amygdala, as well as, from the basal to the central amygdala. This culminates in GABAergic output from the central amygdala to modulatory systems like the dopamine, serotonin, and norepinephrine neurons in the mid and hindbrain, as well as, to the hypothalamus where neuroendocrine system may be modulated (LeDoux, 2008). This can also be gated from the prefrontal cortex via the intercalated nucleus which then inhibits the central amygdala’s output (Figure 29). Reduced emotionality is one of the cardinal symptoms of schizophrenia (World Health Organization, 1992; American
Psychiatric Association, 2013). With the high amount of GABAergic cells in the intercalated nucleus and many of these positive for ErbB4, it is not hard to imagine that over activation of ErbB4 in this area could result in inhibited output from the central amygdala as possibly blunted affect.
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Figure 29. Intercalated Nucleus Role In Blunted Affect And Connectivity Of the Amygdala
(A) Simplified model. Sensory information flows from sensory cortices to lateral amygdala (LA), then passed to basal amygdala (B). This is then relayed to central amygdala (CE). This is gated by the intercalated nucleus (ITC). Prefrontal cortex can stimulated ITC to inhibit CE. CE output goes to dopamine (DA), serotonin (5-HT), and norepinephrine (NE) neurons and to hypothalamus. ( B)
True complexity of the amygdala involves input from various brain regions, feedback from the amygdala, interconnection of all amygdala nuclei, and output to various regions of the brain with feedback. M, medial amygdala.
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4.3 ErbB4 In GABA or Glutamatergic Neurons
Previous reports either claimed or suggested that ErbB4 was expressed in glutamatergic neurons (Garcia et al., 2000; Huang et al., 2000; Ma et al., 2003;
Kwon et al., 2005; Li et al., 2007; Iyengar and Mott, 2008; Barros et al., 2009;
Pitcher et al., 2011). In-vitro studies noted that cultured cortical or hippocampal neurons, that would be mostly glutamatergic, responded to NRG1 treatment with increased neurite outgrowth (Gerecke et al., 2004; Audisio et al., 2012).
However, others have disputed this, claiming that this effect is only observed in
GABAergic interneurons (Krivosheya et al., 2008; Cahill et al., 2012). On the other hand, excitatory spine morphology and behavior are impaired by ErbB4 gene deletion via GFAP::Cre , Nestin::Cre , and Emx1::Cre which are expressed in cells including pyramidal neurons (Barros et al., 2009). It is well documented that NRG1 treatment can inhibit excitatory neuron firing and long term potentiation (LTP) (Huang et al., 2000; Kwon et al., 2005; Pitcher et al., 2008;
Chen et al., 2010; Wen et al., 2010). The mechanism is, however, debated.
Some place ErbB4 on GABAergic neurons suggesting that the effect is secondary to NRG1 enhanced GABA release (Chen et al., 2010; Wen et al.,
2010), while others place ErbB4 on excitatory neurons suggesting a cell autonomous effect (Gu et al., 2005; Kwon et al., 2005; Li et al., 2007; Pitcher et al., 2011).
In support of ErbB4 being expressed primarily in GABAergic neurons, transcripts were found in areas known to be enriched with interneurons and had a similar distribution (Lai and Lemke, 1991; Woo et al., 2007). ErbB4 protein was
145 detected in GAD-positive cells in the hippocampus (Huang et al., 2000; Woo et al.). Some have gone as far as to say that ErbB4 is exclusively expressed in
GABAergic neurons in cortex and hippocampus (Vullhorst et al., 2009; Fazzari et al., 2010; Neddens and Buonanno, 2011; Neddens et al., 2011). Nevertheless, a recent report suggested that ErbB4 protein could be found in some cortical excitatory neurons (Cooper and Koleske, 2014). Therefore, it became critically important to determine exactly in what cells ErbB4 is expressed. Unfortunately, this question cannot be addressed by current antibodies against ErbB4 because of poor specificity and availability.
I attempted to address this problem by utilizing genetic labeling techniques. Mice with an inducible Cre driven by endogenous ErbB4 promoter
(ErbB4::CreERT2 ) were crossed with mice that had tdTomato red fluorescent protein inserted in Rosa26 gene with a floxed stop sequence just before
(Rosa26::LSL-tdTomato ) (Madisen et al., 2010). Once CreERT2 was activated by tamoxifen, the stop sequence in front of tdTomato would be removed and tdTomato expression would ensue, thus filling the cell with the red fluorescent protein. In this way, cells that were expressing ErbB4 at the time of induction were unmistakable from those that were not. The inducible aspect gave me temporal resolution to ask, what cells express ErbB4 at adult and neonatal stages. By crossing the ErbB4-reporter mice with GAD67::GFP mice, that express a green fluorescent protein in GABAergic cells, I was able to access the degree to which ErbB4 was expressed in GABAergic cells. In forebrain regions
Erbb4 was almost always expressed in GABAergic neurons, with ≥ 98% found in
146 cortex and hippocampus. It was noted in the original characterization of
GAD67::GFP mice that not all GABAergic cells were labeled equally and depending on the threshold set, not all were detectable (Tamamaki et al., 2003).
Thus it is possible that 98% may in fact be an underestimate. Regardless, the predominate cellular target of NRG1-ErbB4 signaling in these forebrain areas appears to be GABAergic interneurons.
Forebrain areas are increasingly implicated in schizophrenia, including the amygdala, which is associated with emotional memory (LeDoux, 2008).
Schizophrenics often have reduced emotionality or flattened affect (Tsuang et al.,
2000) and alteration in amygdala activity (Brunet-Gouet and Decety, 2006;
Pinkham et al., 2007; Rasetti et al., 2009). High levels of ErbB4 in amygdala may provide a pathophysiological mechanism of abnormal NRG1-ErbB4 signaling.
GABAergic dysfunction is well documented in schizophrenia (Gonzalez-Burgos et al., 2011; Lewis et al., 2011). Finding ErbB4 highly expressed in GABAergic neurons together with NRG1’s noted ability to promote GABA release and to suppress LTP in a manner that requires ErbB4 (Woo et al., 2007; Chen et al.,
2010; Wen et al., 2010) provides additional insight into the pathophysiological mechanisms that may be in play in those carrying polymorphisms in either NRG1 or ErbB4 . Intriguingly, ErbB4 gene mutation causes schizophrenia-relevant behavior deficits including hyperactivity, impaired prepulse inhibition, abnormal working memory and contextual fear conditioning (Stefansson et al., 2002; Chen et al., 2010; Wen et al., 2010; Shamir et al., 2012; Del Pino et al., 2013).
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4.4 ErbB4 In the Hypothalamus And Neuregulin-1 – ErbB4 Signaling’s
Possible Role In Metabolism
ErbB4-positive cells were enriched in hypothalamic nuclei including posterior hypothalamic, dorsal medial hypothalamic, and paraventricular hypothalamic (PaVH) nuclei. Previous reports had noted ErbB4’s presence in hypothalamus describing its presence as being mostly in astrocytes (Ma et al.,
1999; Prevot et al., 2003; Sharif et al., 2009; Sandau et al., 2011b; Sandau et al.,
2011a). I observed that most ErbB4-positive cells in hypothalamus appeared to have neuronal morphology with approximately half co-localizing with GAD67-
GFP. Unexpectedly, the hybrid reporter mice proved to be invaluable for identifying areas that harbored non-GABAergic ErbB4-positive cells. The PaVH nucleus was one such area. Oxytocin and vasopressin are both known to be expressed in this area (Keverne and Curley, 2004; Lee et al., 2009). ErbB4 was detectable in ~50% of oxytocin-positive neurons in PaVH but never in vasopressin neurons. Oxytocin is involved in social bonding, sexual arousal, orgasm, maternal care, and lactation (Lee et al., 2009). In light of impaired social interaction in schizophrenia (Tsuang et al., 2000), the finding of ErbB4 in oxytocin-expressing neurons suggests a previously unappreciated pathophysiological mechanism of abnormal NRG1-ErbB4 signaling.
ErbB4-positive cell enrichment in hypothalamic nuclei also suggests a role for NRG1-ErbB4 signaling in metabolism and energy homeostasis. POMC,
AgRP, and Kiss1 neurons in the Arcuate nucleus of the hypothalamus sense glucose, insulin, leptin, and ghrelin levels and send appropriate information onto
148 neurons in the PaVH (Sanz et al., 2015). Excitatory neurons in the PaVH then project to parabrachial nucleus (Pb) in the hindbrain. The Pb is also modulated by ventral Raphe serotonin neurons and excitatory input from the nucleus tractus solitarii (NTS) in the hind brain. The final output of the Pb controls feeding and metabolism (Palmiter, 2011). ErbB4-positive cells were enriched in all of these regions (save NTS that was not accessed). These results suggest that NRG1-
ErbB4 signaling may play an important role for energy homeostasis and metabolism.
4.5 Glia Cells Positive For ErbB4 In the Thalamus
In the thalamus ErbB4-positive cells were enriched in the habenula as previously reported (Lai and Lemke, 1991; Steiner et al., 1999; Bruce et al.,
2002; Woo et al., 2007). However, ErbB4-positive cells were remarkably scarce throughout the rest of the thalamus. This did, however, provide a unique opportunity to observe ErbB4-positive cells with non-neuronal morphology. Under closer inspection they were also present in all other subcortical areas. These cells had small soma with many radiating processes. I originally hypothesized these would be oligodendrocytes but they did not co-localize with either Olig2 or myelin basic protein, two markers for oligodendrocytes. I next guessed that they may be astrocytes as previous reports had noted ErbB4 present in cells positive for GFAP (Ma et al., 1999; Prevot et al., 2003; Sharif et al., 2009; Sandau et al.,
2011b; Sandau et al., 2011a). These cells did stain positive for S100β, one
149 marker for astrocytes, but not for GFAP, leading me to conclude they may be non-reactive astrocytes or unidentified oligodendrocytes.
4.6 ErbB4 Is Selectively Expressed In Monoamine Neurons Implicated In
Psychiatric Disorders
ErbB4-positive cells in the midbrain were enriched in monoaminergic nuclei. Previous in-situ studies have noted ErbB4 in presence in dopamine neurons (Abe et al., 2009; Zheng et al., 2009), on the other hand, immunological studies have either concluded that ErbB4 is not present or is expressed at low levels (Kwon et al., 2008; Neddens and Buonanno, 2011). Using the ErbB4- reporter mice I was able to confirm ErbB4 was indeed expressed in dopamine neurons. More strikingly, ErbB4-positive cells were enriched in several Raphe nuclei. Furthermore, these cells were immuno-reactive to the neurotransmitter, indicating they were serotonin neurons. Serotonin has long been implicated in psychiatric disorders including schizophrenia (Laruelle et al., 1993; Ngan et al.,
2000). This identifies a novel cellular target for NRG1-ErbB4 signaling, and adds a layer of complexity to how NRG1 and ErbB4 may be related to the disorder.
Interestingly, ErbB4 was selectively expressed in dopamine and serotonin neurons as norepinephrine neurons were not positive for ErbB4. Norepinephrine neurons are known to be particularly silent during sleep and particularly active during vigilance (Nolte, 2009c). It is possible that NRG1-ErbB4 signaling is restricted to modulatory neurons and not those whose primary purpose is to excite.
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4.7 Limitations Of ErbB4-reporter Mice
Both in-situ and immunohistochemical studies have evidenced ErbB4’s presence in the reticular thalamic nucleus (Lai and Lemke, 1991; Steiner et al.,
1999; Bruce et al., 2002; Woo et al., 2007; Neddens and Buonanno, 2011;
Ahrens et al., 2015), whereas ErbB4-reporter mice revealed little to no positive cells in this area. While immunohistochemical studies were unable to produce convincing evidence of ErbB4’s presence in dopamine neurons, in-situ studies proclaimed ErbB4 mRNA was in most (80-99%) dopamine neurons in midbrains of rodents, monkey and human (Abe et al., 2009; Zheng et al., 2009). However, only 20% of midbrain dopamine neurons expressed tdTomato in ErbB4-reporter mice. It is worth pointing out that the readout of this method depends on activation of the CreERT2 by tamoxifen that is controlled by tamoxifen bioavailability and expression and stability of the tdTomato protein. Staining with an antibody against DsRed, from which tdTomato was derived, did not label any additional cells indicating that the disparity in results was not due to loss of signal
(Figure 30). Thus the major strength of having temporal control may have also been the major limitation of this study.
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Figure 30. DsRed Does Not Label Any Additional Cells
(A) Image of the reticular thalamic nucleus (RTN). GAD67 in green marking the
GABAergic neurons of the RTN. Few ErbB4-positive cells, in red, were present in the RTN. Those that were present seldom co-localized with GAD67. An antibody against DsRed, in blue, labeled the same cells as the ErbB4-reporter did and not any additional cells. ( B) Image of the ventral tegmental area (VTA). In red cells labeled by ErbB4-reporter method. In green, DsRed antibody labeled the same cells but not any additional cells.
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4.8 Neuregulin-1 Enhanced Monoamine Release
NRG1 has been shown to modify the response of neurotransmitters suppressing glutamatergic neuron firing or LTP, or enhancing the release of
GABA and dopamine (Huang et al., 2000; Yurek et al., 2004; Kwon et al., 2005;
Woo et al., 2007; Kwon et al., 2008). Two models have emerged regarding enhanced release of neurotransmitters. One suggests that NRG1 acts on ErbB4 at the synapse to enhance evoked release of GABA (Woo et al., 2007) or spontaneous dopamine release in dopaminergic fibers in the hippocampus
(Kwon et al., 2008). The other model suggests that NRG1 acts on ErbB4 on the soma of dopamine neurons in the SNc to stimulate dopamine release in the striatum (Yurek et al., 2004).
The medial PFC is known to receive projections from norepinephrine, dopamine, and serotonin neurons (Nolte, 2009c). Having confirmed ErbB4’s presence in dopamine and serotonin neurons, I set out to test the idea that
NRG1 could enhance the spontaneous release of dopamine and serotonin without affecting norepinephrine release. Perfusion of NRG1 into medial PFC did induce both dopamine and serotonin release, however, these results were unlike those previously reported for neurotransmitter release. Previous reports had always noted an immediate response and a return to baseline within 60 min.
(Yurek et al., 2004; Woo et al., 2007; Kwon et al., 2008). Through my experiment
I observed that dopamine neurons responded by releasing more dopamine after a short delay of 20 min. and continued through 120 min. More interestingly, serotonin neurons did not respond until 60 min. post perfusion and lasted through
154
140 min. Through 180 min. norepinephrine neurons still had not responded.
Differences in my results and previous reports could be due to different methodology. I perfused NRG1 (3.33 µM x 20 min = ~0.5 µg) over 20 min., while others had either injected NRG1 (10 µg) over 2 min. (Yurek et al., 2004) or perfused NRG1 (1 nM x 5 min = ~4 ng) over 5 min. (Kwon et al., 2008).
The disparity in dopamine and serotonin neurons’ NRG1 enhanced neurotransmitter release raises the question as to why they would respond differently. It may be that NRG1 is acting on ErbB4 at the synapse and a retrograde signal is sent back to the soma (Figure 31A). Because dopamine neurons are closer to medial PFC they would be able to respond quicker than serotonin neurons that are located more caudally. An alternative model is that
NRG1 possibly defuses into the nearby ventricular system and circulates back from the lateral ventricle through the third ventricle then cerebral aqueduct
(Figure 31B). This model however, seems less likely as the VTA and SNc are not nearby the ventricular system whereas the dorsal Raphe is located right under the cerebral aqueduct.
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Figure 31. Models For Delayed Neuregulin-1 Enhanced Monoamine Release
(A) Perfusion of NRG1 into the medial PFC activates ErbB4 at the synapse of serotonin (5-HT) and dopamine (DA) neurons but not norepinephrine (NE) neurons. Intercellular domain of ErbB4 (ICD-ErbB4) is cleaved and transported retrograde for nuclear signaling. ( B) Perfusion of NRG1 into the medial PFC leaks into nearby lateral ventricle (LV). Traveling through the ventricular system
NRG1 activates ErbB4 on the soma of nearby 5-HT neurons. III, 3 rd ventricle; IV,
4th ventricle
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4.9 Translations Implications For Altered Neuregulin-1 – ErbB4 Signaling
Both NRG1 and ErbB4 have been implicated in schizophrenia in human populations (Stefansson et al., 2002; Silberberg et al., 2006). Disruption of either
NRG1 or ErbB4 in mice leads to many behavioral and electrophysiological phenotypes that are relevant to schizophrenia (Stefansson et al., 2002; Chen et al., 2010; Wen et al., 2010; Yin et al., 2013b). Identifying the locations and cellular identity of ErbB4 expressing cells and thus the cellular targets of NRG1-
ErbB4 signaling gives us a richer understanding of the neuronal systems affected by polymorphisms seen in these genes.
This information could be taken into account while designing treatment plans for schizophrenics identified as having possible altered NRG1-ErbB4 signaling. Current treatments target dopamine D2-type receptors and serotonin
5-HT2A receptors. Both dopamine and serotonin neurons express ErbB4 but this treatment fails to account for GABAergic tone that is likely altered in individuals suffering NRG1/ErbB4 relevant schizophrenias. Oxytocin tone may also be altered in these individuals and may account for some of the symptomology.
Future treatments may directly target ErbB4 with small molecules that mimic NRG1 or otherwise activate or inhibit ErbB4 (Mikkelsen et al., 2010). Other treatments may promote, mimic, or inhibit the metalloproteases’ that cleave pro-
NRG1 to initiate or block signaling events depending on the patients’ needs.
NRG1 could also be injected intracerebroventricularly (ICV) (Snodgrass-Belt et al., 2005) or peripherally (Ennequin et al., 2015) to stimulate signaling or a dominate negative version to block signaling.
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In the distant future it may be possible to use genetic editing to correct polymorphisms either in affected individuals or embryonically before the person is born and has had the chance to develop the disorder of schizophrenia. While this notion was inconceivable just a few years before ago, new technologies like
CRISPR/CAS9 (Horvath and Barrangou, 2010; Cong et al., 2013) now make it a real possibility. Recently it was even demonstrated that a non-viable human embryo could be edited using the technology (Liang et al., 2015).
Finally, the true value of the research contained within is beyond the scope of any translational application. The basic knowledge of where in the brain and in what cell type ErbB4 is expressed, as well as, the ability of NRG1 perfusion into PFC to stimulate enhanced dopamine and serotonin release is infinitely more valuable than any single application . For basic knowledge not only adds to our understanding of the natural world it also has infinite applications that are beyond our current understanding.
5. SUMMARY
ErbB4 is widely expressed in the adult brain with obvious region differences in positive cell density. Amygdala followed by cortex and hypothalamus have the highest densities, while thalamus, hindbrain, and cerebellum are among brain regions with the lowest. In the cortex, hippocampus, basal ganglia, and most of amygdala ErbB4-positive cells are almost exclusively GABAergic neurons.
Suggesting GABAergic interneurons are the major target of Neuregulin-1
(NRG1)-ErbB4 signaling in these areas.
In hypothalamus ErbB4-positive cells are sometimes oxytocin neurons.
This identifies a novel cellular target for NRG1-ErbB4 signaling and provides a possible mechanism for some of the psychopathology seen in schizophrenia.
The high levels of ErbB4-positive cells in several hypothalamic nuclei additionally suggest a role for NRG1-ErbB4 signaling in metabolism.
In the midbrain ErbB4-positive cells are dopamine and serotonin neurons but not norepinephrine neurons. This suggests a selective rather than promiscuous targeting for NRG1-ErbB4 signaling. The novel cellular target of serotonin neurons adds a layer of complexity to NRG1 and ErbB4’s involvement in schizophrenia and other psychiatric disorders. Throughout subcortical areas
158
159 non-neuronal ErbB4-positive cells were observed that stained positive for S100β, but not for other glia markers.
Finally, NRG1 perfusion into the prefrontal cortex can enhance both dopamine and serotonin release over a prolonged period with serotonin neurons responding later than dopamine neurons. This result suggests a previously undiscovered mechanism for NRG1 enhanced neurotransmission, namely a retrograde signaling event that could not have been appreciated in other cellular targets with shorter axons such as interneurons.
.
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