Elsevier

Journal of Chemical Neuroanatomy

Volume 52, September 2013, Pages 36-43
Journal of Chemical Neuroanatomy

Organic cation transporter 3 is densely expressed in the intercalated cell groups of the amygdala: Anatomical evidence for a stress hormone-sensitive dopamine clearance system

https://doi.org/10.1016/j.jchemneu.2013.04.007Get rights and content

Highlights

  • The corticosterone-sensitive monoamine transporter OCT3 is expressed in nearly all neurons of the intercalated cell groups of the amygdala.

  • OCT3 is expressed in close proximity to, but not on dopaminergic terminals in the intercalated cell groups.

  • OCT3 is expressed in close proximity to dopamine D1 receptors in the intercalated cell groups.

  • OCT3 is proposed to represent a previously undescribed dopamine clearance mechanism in the intercalated cell groups.

Abstract

The intercalated cell groups of the amygdala (ITCs) are clusters of GABAergic neurons which exert powerful modulatory control of amygdala output, and are thought to play key roles in the extinction of conditioned fear responses. Dopamine, acting through D1 receptors, inhibits ITC neuronal activity, an action that has the potential to disinhibit amygdala activity, leading to changes in behavioral responses. Dopaminergic neurotransmission in the ITC occurs through a combination of synaptic and volume transmission. Thus, mechanisms, including transport mechanisms, that regulate extracellular dopamine concentrations in the ITC, are likely to be important determinants of amygdala function. We have recently demonstrated the expression of organic cation transporter 3 (OCT3), a high-capacity transporter for dopamine and other monoamines, throughout the rat brain. In this study, we used immunohistochemical and immunofluorescence techniques to examine the distribution of OCT3 in the ITC, to identify the phenotype of OCT3-expressing cells, and to describe the spatial relationships of OCT3 to dopaminergic terminals and dopamine D1 receptors in these areas. We observed high densities of OCT3-immunoreactive perikarya and punctae throughout the D1 receptor-rich main, anterior and paracapsular ITCs, in contrast with the basolateral amygdala, where OCT3 immunoreactive perikarya and puncta were observed at much lower density. OCT3-immunoreactive perikarya in the ITC were identified as neurons. Tyrosine hydroxylase-immunoreactive fibers in the ITC were immunonegative for OCT3, though OCT3-immunoreactive punctae were observed in close proximity to TH+ terminals. Punctate OCT3-immunoreactivity in the ITCs was observed in very close proximity (<1 μm) to D1 receptor immunoreactivity. These anatomical data are consistent with the hypothesis that OCT3 plays a central role in regulating dopaminergic neurotransmission in the ITC, and that it represents a post- or peri-synaptic dopamine clearance mechanism. Inhibition of OCT3-mediated transport by corticosterone may represent a mechanism by which acute stress alters dopaminergic neurotransmission in the amygdala, leading to alterations in fear and anxiety-like behavior.

Introduction

The amygdala is a critical component of the neural circuitry mediating anxiety and fear (Davis et al., 1994, Ledoux, 2000). Effective regulation of anxiety and fear-related behaviors requires strict control over the activity and excitability of neurons in the basolateral amygdala (BLA) and the central amygdala (CeA), so that anxiety and fear responses are suppressed under most conditions, but can be rapidly expressed when necessary. This regulation is accomplished in part by a complex inhibitory network surrounding the amygdala which, driven primarily by cortical projections, suppresses BLA and CeA activity and thus prevents inappropriate expression of fear and anxiety responses (Davis and Myers, 2002, Quirk and Gehlert, 2003, Sanders and Shekhar, 1995). When these responses are appropriate, however, inhibitory control of amygdala output must be released. Recent studies have demonstrated that the dopaminergic system plays a critical role in releasing cortical inhibition of amygdala activity, and thus is a potent regulator of fear and anxiety responses (de la Mora et al., 2010, Fadok et al., 2009, Guarraci et al., 1999). Acting primarily through D1 receptors, dopamine increases the activity of BLA pyramidal neurons by attenuating inhibitory influence from the infralimbic prefrontal cortex (PFC) and enhancing sensory cortical inputs, resulting in overall enhancement of BLA-mediated behaviors (Greba et al., 2001, Lamont and Kokkinidis, 1998, Rosenkranz and Grace, 2001). Thus, D1 receptors are thought to function as a switch, facilitating the transition of the BLA from a PFC-controlled, relatively inhibited state to a disinhibited, more excitable state (Rosenkranz and Grace, 2002). Recent studies suggest that the effects of dopamine on amydala activity result in large part from its actions on neurons in the intercalated cell groups of the amygdala (ITC) (Marowsky et al., 2005).

The ITCs consist of dense clusters of D1 receptor-expressing small-to-medium-sized GABAergic neurons surrounding the BLA, and are subdivided into the main (IM), anterior (IA) and lateral and medial paracapsular (Ilp and Imp, respectively) islands (Millhouse, 1986). ITC neurons receive prominent excitatory inputs from the PFC (Berretta et al., 2005, Quirk et al., 2003, Sesack et al., 1989, Vertes, 2004) and send projections to the central and basolateral amygdaloid nuclei, as well as to adjacent ITC cell groups (Royer et al., 1999, Royer et al., 2000). ITC neurons are activated during fear expression, extinction training and extinction retrieval (Busti et al., 2011), and in response to pharmacological activation of infralimbic PFC neurons (Berretta et al., 2005). Recent studies have demonstrated that the intercalated cell groups are functionally heterogeneous, and suggest that they form a complex interconnected network that controls the sensitivity of the amygdala to afferent neuronal signals and regulates communication between distinct nuclei within the amygdala. These studies suggest that cells in Imp and IM regulate the flow of neuronal signals from the BLA to the central nucleus (Royer et al., 1999, Manko et al., 2011), while Ilp neurons regulate PFC-to-amygdala signals (Marowsky et al., 2005). Neurons in the IA may modulate communication between right and left amygdalae (Marcellino et al., 2012). In addition, the ITCs are functionally and anatomically interconnected, such that activation of one intercalated cell group may result in inhibition of another. Stimulation of IM, Ilp and Imp neurons by infralimbic PFC projections results in inhibition of BLA and CeA projection neurons (Marowsky et al., 2005, Amir et al., 2011), and lesions of Imp neurons result in increases in anxiety and fear, and deficits in extinction of conditioned fear (Pape and Pare, 2010, Likhtik et al., 2008).

Intercalated cell groups receive dense dopaminergic projections from the VTA (Asan, 1998, Freedman and Cassell, 1994, Moore and Bloom, 1978), and express the highest concentration of dopamine D1 receptors in the mammalian amygdala (Fuxe et al., 2003). Activation of D1 receptors hyperpolarizes and decreases the firing rates of GABAergic neurons in both the Imp and the IM (Marowsky et al., 2005, Manko et al., 2011). In the Imp, this effect is mediated by D1-receptor-induced activation of G protein-coupled inwardly rectifying potassium (GIRK) channels, decreasing the sensitivity of ITC neurons to PFC stimulation, and resulting in disinhibition of BLA and CeA activity (Marowsky et al., 2005). Thus extracellular dopamine, by regulating ITC-mediated inhibitory tone, is a critical determinant of amygdala excitability and plasticity. Stimuli, including stress-related stimuli, which increase dopamine concentrations in these cell groups, would lead to altered sensitivity of BLA neurons to excitatory signals.

Neuroanatomical studies of the ITC indicate that a substantial portion (up to 50%) of dopaminergic neurotransmission within these areas occurs via volume transmission (Fuxe et al., 2003, Fuxe et al., 2005, Marcellino et al., 2012). Thus, mechanisms, including presynaptic or postsynaptic transport, that regulate extracellular dopamine concentration are likely to be important determinants of amygdala function. Previous studies have demonstrated that the pattern of dopamine transporter (DAT) immunoreactivity in the amygdala overlaps with that of tyrosine hydroxylase (TH) and is heterogeneous, with densities within the ITCs approaching the high levels seen in the striatum, moderate densities in the central lateral nucleus, and low densities within the basolateral and lateral nuclei (Revay et al., 1996, Ciliax et al., 1995). Recent descriptions of TH immunostaining have described a spatial “mismatch” between these dopaminergic terminals and D1 receptors in the ITC, with distances of 1 μm or more separating D1 receptors from TH-immunoreactive terminals, suggesting that other clearance mechanisms besides the presynaptic DAT may be involved in regulating dopaminergic neurotransmission in these areas (Marcellino et al., 2012, Fuxe et al., 2003).

We have recently described the expression in the amygdala of an additional dopamine clearance mechanism, organic cation transporter 3 (OCT3), with particular enrichment in dense clusters of small cells surrounding the BLA (Gasser et al., 2009). In contrast to the DAT, OCT3 has higher capacity and lower affinity for dopamine, is sodium-independent, and has the capacity to transport norepinephrine, serotonin, and other monoamines (Duan and Wang, 2010, Grundemann et al., 1998, Grundemann et al., 1999). Interestingly, OCT3-mediated transport is directly and acutely inhibited by the stress hormone corticosterone (Grundemann et al., 1998). Thus, OCT3 may represent a stress-sensitive dopamine clearance mechanism, and may act within the amygdala to control extracellular dopamine concentrations. Because of the profound functional implications of high-density expression of a previously uncharacterized dopamine transporter in this important brain region, and in order to understand the contribution of OCT3 to the regulation of dopaminergic neurotransmission, we sought in this study to more fully describe the distribution of OCT3-immunoreactivity in the ITC, to examine its relationship to D1 receptors and dopaminergic terminals in the ITC, and to identify the phenotype(s) of OCT3-expressing cells.

Section snippets

Materials and methods

Male Sprague Dawley rats (Harlan Laboratories, Inc., St. Louis, MO, USA), weighing 275–325 g, were housed individually in a temperature- and humidity-controlled, AAALAC-accredited vivarium under a 12 h/12 h light–dark cycle (lights on at 0700 h) with ad libitum access to food and water. Housing conditions and experimental protocols approved by the Marquette University Institutional Animal Care and Use Committee, and were carried out in accordance with the NIH Guide for the Care and Use of

OCT3 immunostaining in the intercalated cell groups: comparison with DA D1r immunostaining

Immunohistochemical localization revealed a consistent pattern of OCT3-like immunoreactivity in the BLA and intercalated cell groups in the brains of each rat (n = 4). OCT3-like immunoreactivity was seen as a brown reaction product concentrated in perikarya, with diffuse punctate brown reaction product visible at higher magnification (Fig. 1A,C). As previously reported, this staining pattern was not observed in tissue sections incubated in the absence of primary antibody (data not shown).

Discussion

These studies provide anatomical evidence that OCT3, a high-capacity transporter for dopamine and other monoamines (Grundemann et al., 1998), may play a prominent role in controlling dopaminergic neurotransmission in the intercalated cell masses of the amygdala. The dense expression of OCT3, its close proximity to dopamine D1 receptors, and its relationship to catecholaminergic terminals, most of which are likely to be dopaminergic terminals (Marcellino et al., 2012), suggest that OCT3-mediated

Conflicts of interest

The authors disclose that they have no actual or potential conflicts of interest related to this work.

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