Elsevier

Brain Research

Volume 1332, 21 May 2010, Pages 48-56
Brain Research

Research Report
Limited convergence of rhinal cortical and dopaminergic inputs in the rat basolateral amygdala: An ultrastructural analysis

https://doi.org/10.1016/j.brainres.2010.03.062Get rights and content

Abstract

The basolateral nuclear complex of the amygdala (BLC) receives robust sensory inputs from the rhinal cortices (RCx) that are important for the generation of emotional behavior. The BLC is also one of the main targets of the mesolimbic dopamine (DA) system. DA potentiates cortical sensory inputs to the BLC, which leads to an increase in the excitability of BLC pyramidal cells. These findings suggest that there may be convergence of RCx and DA inputs onto the dendrites of pyramidal cells in the BLC. In the present study we used dual-labeling immunohistochemistry and anterograde tract-tracing at the ultrastructural level to test this hypothesis in the rat brain. RCx axons were labeled by Phaseolus vulgaris leucoagglutinin (PHA-L) injections, whereas tyrosine hydroxylase (TH) was used as a marker for DA axons. The extent of convergence of these axons was analyzed in the posterior subdivision of the basolateral nucleus (BLp), which is densely innervated by both inputs. RCx synapses were asymmetrical and mainly contacted dendritic spines (86.4%) and dendritic shafts (12.1%). TH-positive (TH+) terminals also mainly formed synapses (symmetrical) and appositions with spines and shafts of dendrites. However, ultrastructural analysis found a very low percentage of RCx terminals converging with DA terminals onto unlabeled dendrites (9.4%) and axons (7.5 %), or exhibiting direct contacts with TH+ terminals (3.8%). These findings suggest that the association of specific behaviorally salient sensory stimuli with dopamine release in the BLC is not dependent on a point-to-point spatial relationship of cortical and DA inputs.

Introduction

Activation of the basolateral nuclear complex of the amygdala (BLC) by sensory cortical inputs is important for the generation of emotional behavior (Armony et al., 1997, Campeau & Davis, 1995, LeDoux et al., 1990, Uwano et al., 1995). In contrast, activation of medial prefrontal cortical (mPFC) inputs to the BLC suppresses many of these amygdala-dependent behaviors (Dias et al., 1996, Morgan & LeDoux, 1995). The entorhinal and perirhinal cortices (i.e. the rhinal cortices, RCx) receive highly processed sensory information from both unimodal and polymodal areas (Suzuki & Amaral, 1994, Burwell et al., 1995, Kerr et al., 2007, Furtak et al., 2007). As part of the medial temporal lobe memory system (Squire and Zola-Morgan, 1991), the RCx integrates and transfers neocortical information to the hippocampus (Burwell et al., 1995) and amygdala (Insausti et al., 1987, Russchen, 1982, Ottersen, 1982). The perirhinal cortex and the lateral entorhinal area provide robust inputs to the lateral aspect of the basolateral nucleus, including the posterior subdivision of the basolateral nucleus (BLp) (Shi & Cassell, 1999, McDonald & Mascagni, 1997, McDonald, 1998). RCx-BLC circuits are involved in the coordination of emotional responses including autonomic, endocrine and behavioral components (Aggleton, 1993, Aggleton & Mishkin, 1986) and are important in driving fast oscillatory activity during learning (Bauer et al., 2007, Collins et al., 2001). RCx-BLC connections may be important for linking declarative memory with implicit emotional memory (Suzuki, 1996).

The amygdala is also one of the main targets of the mesolimbic dopamine (DA) system (Sadikot & Parent, 1990, Fallon & Ciofi, 1992, Asan, 1997). Dopaminergic inputs to the BLC arise in the ventral tegmental area and substantia nigra (Fallon and Ciofi, 1992). Release of DA during stress is much higher in the BLC than in other targets of the mesolimbic DA system (Coco et al., 1992, Inglis & Moghaddam, 1999), and dopaminergic projections to the BLC are critical for fear conditioning and other aversive behaviors (Pezze & Feldon, 2004, Lalumiere et al., 2004). The DA system also plays an important role in modulating cortical input into the BLC. During fear conditioning, DA reduces mPFC input and potentiates sensory cortical input to the amygdala, which subsequently leads to an increase in the activity of BLC pyramidal cells (Rosenkranz & Grace, 2001, Rosenkranz & Grace, 2002a, Rosenkranz & Grace, 2002b).

The main postsynaptic targets of DA inputs to the BLC are distal dendrites and spines of pyramidal cells that often receive other asymmetrical (excitatory) inputs (Asan, 1997, Pinard et al., 2008, Muller et al., 2009). One source of excitatory inputs to the spines and distal dendrites of BLC pyramidal cells is the cerebral cortex (Hall, 1972, Smith & Paré, 1994, Brinley-Reed et al., 1995, Farb & LeDoux, 1999, Smith et al., 2000). Ultrastructural analysis of mPFC projections to the BLC has demonstrated that there is limited convergence of mPFC and DA terminals onto the same postsynaptic neuron (Pinto and Sesack, 2008). However, there have been no studies examining the convergence of sensory cortical and DA terminals onto the same neurons in the BLC. The present study combined tract-tracing and immunocytochemical methods at the ultrastructural level to characterize the patterns of synaptic connectivity between RCx and DA terminals in the BLp. The BLp was chosen because this area receives a dense innervation by both RCx and DA fibers. Given the fact that DA potentiates sensory cortical inputs, and the importance of both sensory cortical input and dopaminergic input in modulating pyramidal cell activity in the BLC (Rosenkranz and Grace, 1999), we hypothesized that RCx terminals and DA terminals would frequently converge on the same postsynaptic structure in the BLp.

Section snippets

Results

Bilateral PHA-L injections into the RCx in 4 rats produced 6 injection sites that were confined to the perirhinal and dorsolateral entorhinal cortex (Fig. 1A). The pattern of PHA-L -labeled fibers in the amygdala was very similar to that seen in previous anterograde studies of these cortical areas (Shi & Cassell, 1999, McDonald & Mascagni, 1997, McDonald, 1998). Thus, in sections processed using Very Intense Purple (PUR) as a chromogen (Fig. 1C), or in adjacent sections using nickel-enhanced

Rhinal cortical inputs to the BLC

The present study is the first investigation to study the possible convergence of RCx and DA terminals in the rat BLC. Our light microscopic observations are consistent with earlier studies of the RCx projections to the BLC (McDonald, 1998, Shi & Cassell, 1999). These previous investigations found that the projections to the lateral BLp arise mainly from the dorsolateral entorhinal cortex and the ventral, but not the dorsal, subregion of the perirhinal cortex. Our electron microscopic findings

PHA-L injection and tissue preparation

These dual-labeling EM studies combined PHA-L anterograde tract-tracing (to label RCx inputs) with immunohistochemistry for tyrosine hydroxylase (TH; to label dopaminergic terminals). Four adult, male Sprague–Dawley rats (Harlan, Indianapolis, IN) received bilateral stereotaxic injections of Phaseolus vulgaris leucoagglutinin (PHA-L; 2.5% dissolved in 10 mM phosphate buffer, pH 8.0) into the RCx. Rats were anesthetized and placed into a stereotaxic head holder. Coordinates for cortical areas

Acknowledgment

This work was supported by NIH Grant R01-DA027305.

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