Sarina M. Rodrigues

Oxytocin receptor genetic variation relates to empathy and stress reactivity in humans

Oxytocin, a peptide that functions as both a hormone and neurotransmitter, has broad influences on social and emotional processing throughout the body and the brain. In this study, we tested how a polymorphism (rs53576) of the oxytocin receptor relates to two key social processes related to oxytocin: empathy and stress reactivity. Compared with individuals homozygous for the G allele of rs53576 (GG), individuals with one or two copies of the A allele (AG/AA) exhibited lower behavioral and dispositional empathy, as measured by the “Reading the Mind in the Eyes” Test and an other-oriented empathy scale. Furthermore, AA/AG individuals displayed higher physiological and dispositional stress reactivity than GG individuals, as determined by heart rate response during a startle anticipation task and an affective reactivity scale. Our results provide evidence of how a naturally occurring genetic variation of the oxytocin receptor relates to both empathy and stress profiles.

Molecular Mechanisms Underlying Emotional Learning and Memory in the Lateral Amygdala

Fear conditioning is a valuable behavioral paradigm for studying the neural basis of emotional learning and memory. The lateral nucleus of the amygdala (LA) is a crucial site of neural changes that occur during fear conditioning. Pharmacological manipulations of the LA, strategically timed with respect to training and testing, have shed light on the molecular events that mediate the acquisition of fear associations and the formation and maintenance of long-term memories of those associations. Similar mechanisms have been found to underlie long-term potentiation (LTP) in LA, an artificial means of inducing synaptic plasticity and a physiological model of learning and memory. Thus, LTP-like changes in synaptic plasticity may underlie fear conditioning. Given that the neural circuit underlying fear conditioning has been implicated in emotional disorders in humans, the molecular mechanisms of fear conditioning are potential targets for psychotherapeutic drug development.

The Influence of Stress Hormones on Fear Circuitry

Fear arousal, initiated by an environmental threat, leads to activation of the stress response, a state of alarm that promotes an array of autonomic and endocrine changes designed to aid self-preservation. The stress response includes the release of glucocorticoids from the adrenal cortex and catecholamines from the adrenal medulla and sympathetic nerves. These stress hormones, in turn, provide feedback to the brain and influence neural structures that control emotion and cognition. To illustrate this influence, we focus on how it impacts fear conditioning, a behavioral paradigm widely used to study the neural mechanisms underlying the acquisition, expression, consolidation, reconsolidation, and extinction of emotional memories. We also discuss how stress and the endocrine mediators of the stress response influence the morphological and electrophysiological properties of neurons in brain areas that are crucial for fear-conditioning processes, including the amygdala, hippocampus, and prefrontal cortex. The information in this review illuminates the behavioral and cellular events that underlie the feedforward and feedback networks that mediate states of fear and stress and their interaction in the brain.

Pavlovian Fear Conditioning Regulates Thr286 Autophosphorylation of Ca2+/Calmodulin-Dependent Protein Kinase II at Lateral Amygdala Synapses

Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays a critical role in synaptic plasticity and memory formation in a variety of learning systems and species. The present experiments examined the role of CaMKII in the circuitry underlying pavlovian fear conditioning. First, we reveal by immunocytochemical and tract-tracing methods that αCaMKII is postsynaptic to auditory thalamic inputs and colocalized with the NR2B subunit of the NMDA receptor. Furthermore, we show that fear conditioning results in an increase of the autophosphorylated (active) form of αCaMKII in lateral amygdala (LA) spines. Next, we demonstrate that intra-amygdala infusion of a CaMK inhibitor, 1-[NO-bis-1,5-isoquinolinesulfonyl]-N-methyl-l-tyrosyl-4-phenylpiperazine, KN-62, dose-dependently impairs the acquisition, but not the expression, of auditory and contextual fear conditioning. Finally, in electrophysiological experiments, we demonstrate that an NMDA receptor-dependent form of long-term potentiation at thalamic input synapses to the LA is impaired by bath application of KN-62 in vitro. Together, the results of these experiments provide the first comprehensive view of the role of CaMKII in the amygdala during fear conditioning.

Fear conditioning drives profilin into amygdala dendritic spines

Changes in spine morphology may underlie memory formation, but the molecular mechanisms that subserve such alterations are poorly understood. Here we show that fear conditioning in rats leads to the movement of profilin, an actin polymerization–regulatory protein, into dendritic spines in the lateral amygdala and that these spines undergo enlargements in their postsynaptic densities (PSDs). A greater proportion of profilin-containing spines with enlarged PSDs could contribute to the enhancement of associatively induced synaptic responses in the lateral amygdala following fear learning.

Memory consolidation of Pavlovian fear conditioning requires nitric oxide signaling in the lateral amygdala

Nitric oxide (NO) has been widely implicated in synaptic plasticity and memory formation. In studies of long‐term potentiation (LTP), NO is thought to serve as a ‘retrograde messenger’ that contributes to presynaptic aspects of LTP expression. In this study, we examined the role of NO signaling in Pavlovian fear conditioning. We first show that neuronal nitric oxide synthase is localized in the lateral nucleus of the amygdala (LA), a critical site of plasticity in fear conditioning. We next show that NO signaling is required for LTP at thalamic inputs to the LA and for the long‐term consolidation of auditory fear conditioning. Collectively, the findings suggest that NO signaling is an important component of memory formation of auditory fear conditioning, possibly as a retrograde signal that participates in presynaptic aspects of plasticity in the LA.

Distribution of NMDA and AMPA receptor subunits at thalamo-amygdaloid dendritic spines.

Synapses onto dendritic spines in the lateral amygdala formed by afferents from the auditory thalamus represent a site of plasticity in Pavlovian fear conditioning. Previous work has demonstrated that thalamic afferents synapse onto LA spines expressing glutamate receptor (GluR) subunits, but the GluR subunit distribution at the synapse and within the cytoplasm has not been characterized. Therefore, we performed a quantitative analysis for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits GluR2 and GluR3 and N-methyl-D-aspartate (NMDA) receptor subunits NR1 and NR2B by combining anterograde labeling of thalamo-amygdaloid afferents with postembedding immunoelectron microscopy for the GluRs in adult rats. A high percentage of thalamo-amygdaloid spines was immunoreactive for GluR2 (80%), GluR3 (83%), and NR1 (83%), while a smaller proportion of spines expressed NR2B (59%). To compare across the various subunits, the cytoplasmic to synaptic ratios of GluRs were measured within thalamo-amygdaloid spines. Analyses revealed that the cytoplasmic pool of GluR2 receptors was twice as large compared to the GluR3, NR1, and NR2B subunits. Our data also show that in the adult brain, the NR2B subunit is expressed in the majority of in thalamo-amygdaloid spines and that within these spines, the various GluRs are differentially distributed between synaptic and non-synaptic sites. The prevalence of the NR2B subunit in thalamo-amygdaloid spines provides morphological evidence supporting its role in the fear conditioning circuit while the differential distribution of the GluR subtypes may reflect distinct roles for their involvement in this circuitry and synaptic plasticity.

Phosphorylation of ERK/MAP kinase is required for long-term potentiation in anatomically restricted regions of the lateral amygdala in vivo

We have previously shown that the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/ MAPK) is transiently activated in anatomically restricted regions of the lateral amygdala (LA) following Pavlovian fear conditioning and that blockade of ERK/MAPK activation in the LA impairs both fear memory consolidation and long-term potentiation (LTP) in the amygdala, in vitro. The present experiments evaluated the role of the ERK/MAPK signaling cascade in LTP at thalamo-LA input synapses, in vivo. We first show that ERK/MAPK is transiently activated/phosphorylated in the LA at 5 min, but not 15 or 60 min, after high-frequency, but not low-frequency, stimulation of the auditory thalamus. ERK activation induced by LTP-inducing stimulation was anatomically restricted to the same regions of the LA previously shown to exhibit ERK regulation following fear conditioning. We next show that intra-LA infusion of U0126, an inhibitor of ERK/MAPK activation, impairs LTP at thalamo-LA input synapses. Collectively, results demonstrate that ERK/MAPK activation is necessary for synaptic plasticity in anatomically defined regions of the LA, in vivo.

Use of Electron Microscopy in the Detection of Adrenergic Receptors

1.1. Physiological Function of Adrenergic Receptors Adrenergic receptors (ARs) belong to a superfamily of the G-proteincoupled receptors and are categorized by their binding to endogenously occurring catecholamines, i.e., norepinephrine and epinephrine. Adrenergic receptors are classified into three groups (α1-, α2, and β-ARs), each of which is further divided into three subtypes. The α1-(α1A-, α1B-, and α1D-ARs) couple with Gq family of G-proteins (G11, G14, G15, and G16) and result in activation of phospholipase C-βs that liberate two second messengers, diacylglycerol and inositol-l,4,5-trisphosphate. The three subtypes of α2ARs are designated α2A-, α2B-, and α2CcAR. On binding with agonists, α2-AR inhibit adenylyl cyclase and calcium channels, but activate potassium channels through coupling to the Gi family of G-proteins (Gi1, Gi2, Gi3, and G0). Finally, the three groups of β-AR are designated β1-, β2-, and β3-AR: these increase the intracellular cAMP content by activating Gs, which is coupled to the enzyme, adenylyl cyclase (1). Functions of the adrenergic receptors in vitro and in vivo have been analyzed mostly by administrating subtype-selective agonists or antagonists (2). However, although ligands specific for the three major adrenergic receptor types are available and have yielded much useful information, most of the ligands currently available do not exhibit sufficient specificity for discriminating among the subtypes (e.g., the A-subtype of α2-AR). Thus, an alternative approch for identifying the function of the subtypes has been to knock out the gene encoding for the particular receptor subtype. This approach has not always been met with success either, probably because other subtypes of catecholaminergic receptors compensate for the knockout. This is one reason many of the advances in our knowledge about the catecholaminergic receptor subtypes are derived from immunocytochemistry. For brain research, in particular, the immunocytochemical approach has been useful. This is because brain function depends critically on the connectivity formed among neurons. Thus, the effect of catecholamines within brain could differ greatly depending on the site of action of the neurotransmitter and on the receptor subtype located near the release of the neurotransmitter. The site of action of catecholamines can vary by region (e.g., visual vs auditory vs multimodal pathways), cell type within the region (e.g., neurons using excitatory transmitter for projecting long distances vs those using an inhibitory transmitter for local circuits or nonneuronal cells, such as astrocytes), and by the subcellular compartment (dendritic shafts, where primarily inhibitory inputs from other neurons are received, vs dendritic spines, where primarily excitatory inputs are received, or in axons, where outputs to other neurons are propagated and transmitted via release of neurotransmitters). For example, our study using an antiserum capable of selectively recognizing the A subtype of α2-ARs revealed that these occur presynaptically (Fig. 1A, B), some of which were positively identified as noradrenergic axon terminals (3). This result was expected, since earlier physiological studies had shown that α2-ARs operate as autoreceptors, inhibiting release of norepinephrine or epinephrine (4). However, we also observed that these receptors occur in noncatecholaminergic axon terminals, indicating that these may also operate as heteroreceptors regulating the release of transmitters other than norepinephrine and epinephrine. Furthermore, this receptor has been observed postsynaptically within the cerebral cortex (5,6; Fig. 1) and the hippocampus (7), even though electrophysiological studies have indicated a lack of α2-AR mediated postsynaptic effects in these forebrain structures (8). Differences in findings such as these indicate that α2-AR in these structures, unlike those in the brainstem, may activate intracellular second messenger cascades without activating potassium channels. Fig. 1 EMs obtained from the monkey prefrontal cortex immunolabeled using the α2A-AR antiserum and HRP-DAB as the immunolabel. (A) The receptor occurs directly over the presynaptic plasma membrane of a labeled terminal (small arrow in LT) forming a synaptic ... Similarly, a series of studies using antisera directed against distinct domains of β-ARs have revealed interesting differences in the receptor’s conformation across developmental states and cell types within intact cerebral cortical tissue. The first polyclonal antiserum that became available for ultrastructural studies was raised by Joh, using the antigen harvested from frog erythrocyte membranes by Strader and colleagues (9). This antiserum yielded immunolabeling of various portions of neurons, including perikarya and axons, but primarily distal dendrites. Astrocytic processes also were immunolabeled using this antiserum (9,10). In sharp contrast to this result, it was observed that polyclonal antisera and monoclonal antibodies (MAbs) directed against the third intracellular loop region yielded immunolabeling primarily of perikaryal regions of neurons (11), although distal dendrites, spines (11), and axons including presynaptic portions of axons (12), also were immunoreactive. Finally, another polyclonal antiserum directed against the C-terminus of β-ARs recognized primarily astrocytic processes in adulthood (13-16; Fig. 2) but also immunolabeled the earliest-formed synapses within neonatal cortices (17; Fig. 3). Each of these immunolabeling patterns was confirmed to be specific by showing abolishment of antigenicity following preadsorption of the primary antibodies (see Notes 1 and 2). Future studies that examine the relationship of β-ARs to the molecules known to interact with them, such as β-arrestin, β-AR kinase, and Gs proteins, under physiologically specified conditions promise to provide detailed knowledge required for understanding the dynamic regulation of cell physiology by epinephrine and norepinephrine. Fig. 2 EMs obtained from serially collected ultrathin sections of tissue immunolabeled dually for the catecholaminergic terminal (CT) marker, tyrosine hydroxylase, and the C-terminus of β-ARs are identified by the HRP-DAB label. β-ARs occur in ... Fig. 3 EMs obtained from postnatal d 10 rat visual cortex, showing β-AR immunoreactivity using HRP-DAB label and an antiserum directed against the C-terminus of the receptor. (A) A dendritic shaft receiving two synaptic inputs from unlabed axon terminals ... Immunocytochemistry has also been useful for studying the subcellular compartmentation of different receptor subtypes that are coexpressed within single cells. For example, adipocytes and heart cells express both the βl- and β3-AR-subtypes of β-ARs. Interestingly, however, such coexpression does not allow for functional compensation, even though both subtypes are coupled to Gs and adenylyl cyclase (18,19). It is possible that differential localization of the receptors within each cell, i.e., compartmentation, influences the response, since signaling molecules and second messengers are not expected to diffuse freely within cells. In another example, Jurevicus and Fischmeister (20) reported the functional compartment of the β1-AR-mediated cAMP accumulation that is important for increase of calcium current through L-type calcium channel in heart, indicating that the β1-AR, but not the β3-AR, was closely associated with the effector molecule. In another example, it has been reported that the α2C- and α1A-ARs mainly localize intracellularly (21,22). The compartmentation can facilitate efficient signal transduction from the receptor to cellular response and avoid unfavorable responses. In cells as polarized as neurons and glia, precise knowledge about receptor localization becomes ever more important, as diffusion of second messengers become restricted to single dendritic spines, dendritic shafts, or axon varicosities. The cellular mechanism by which receptors, G-proteins, and effector molecules become properly localized is yet unknown. Clearly, elucidation of such cellular mechanism requires precise knowledge about the subcellular localization of the receptor and related elements, for which specific antibodies that recognize the receptors are required.