Elsevier

Neuroscience

Volume 290, 2 April 2015, Pages 227-242
Neuroscience

GABAergic somatostatin-immunoreactive neurons in the amygdala project to the entorhinal cortex

https://doi.org/10.1016/j.neuroscience.2015.01.028Get rights and content

Highlights

  • Most amygdalar neurons labeled by Fluorogold injections into the entorhinal cortex are located in the basolateral amygdala.

  • About 1–5% of these amygdalar projection neurons are nonpyramidal neurons containing somatostatin and/or neuropeptide Y.

  • Slightly more than half of these long-range nonpyramidal neurons (LRNP neurons) express GAD or the m2 muscarinic receptor.

  • No LRNP neurons with entorhinal projections express parvalbumin or vasoactive intestinal peptide.

  • No amygdalar LRNP neurons project to the prefrontal cortex.

Abstract

The entorhinal cortex and other hippocampal and parahippocampal cortices are interconnected by a small number of GABAergic nonpyramidal neurons in addition to glutamatergic pyramidal cells. Since the cortical and basolateral amygdalar nuclei have cortex-like cell types and have robust projections to the entorhinal cortex, we hypothesized that a small number of amygdalar GABAergic nonpyramidal neurons might participate in amygdalo-entorhinal projections. To test this hypothesis we combined Fluorogold (FG) retrograde tract tracing with immunohistochemistry for the amygdalar nonpyramidal cell markers glutamic acid decarboxylase (GAD), parvalbumin (PV), somatostatin (SOM), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), and the m2 muscarinic cholinergic receptor (M2R). Injections of FG into the rat entorhinal cortex labeled numerous neurons that were mainly located in the cortical and basolateral nuclei of the amygdala. Although most of these amygdalar FG+ neurons labeled by entorhinal injections were large pyramidal cells, 1–5% were smaller long-range nonpyramidal neurons (LRNP neurons) that expressed SOM, or both SOM and NPY. No amygdalar FG+ neurons in these cases were PV+ or VIP+. Cell counts revealed that LRNP neurons labeled by injections into the entorhinal cortex constituted about 10–20% of the total SOM+ population, and 20–40% of the total NPY population in portions of the lateral amygdalar nucleus that exhibited a high density of FG+ neurons. Sixty-two percent of amygdalar FG+/SOM+ neurons were GAD+, and 51% were M2R+. Since GABAergic projection neurons typically have low perikaryal levels of GABAergic markers, it is actually possible that most or all of the amygdalar LRNP neurons are GABAergic. Like GABAergic LRNP neurons in hippocampal/parahippocampal regions, amygdalar LRNP neurons that project to the entorhinal cortex are most likely involved in synchronizing oscillatory activity between the two regions. These oscillations could entrain synchronous firing of amygdalar and entorhinal pyramidal neurons, thus facilitating functional interactions between them, including synaptic plasticity.

Introduction

The parahippocampal region, consisting of the entorhinal and perirhinal cortices, is an important part of the medial temporal lobe memory system (Squire and Zola-Morgan, 1991). It relays sensory information from the neocortex to the hippocampus and then transmits hippocampal outputs back to the neocortex for memory storage. The entorhinal cortex, which projects directly to the hippocampus, receives robust inputs from the amygdala that have been shown to be involved in fear conditioning and the facilitation of long-term memory consolidation by emotional arousal (Roesler et al., 2002, Majak and Pitkanen, 2003, McIntyre et al., 2012). Various aspects of fear learning and memory involve synchronization of theta activity in the basolateral amygdala and dorsal hippocampus (Seidenbecher et al., 2003, Pape et al., 2005, Narayanan et al., 2007a, Narayanan et al., 2007b). Since the dorsal hippocampus and basolateral amygdala are not directly interconnected, synchronization of theta activity between these structures may involve a relay in the entorhinal cortex (Mizuseki et al., 2009). These coherent oscillations produce recurring time windows that facilitate synaptic interactions, including synaptic plasticity involved in mnemonic function (Paré et al., 2002). In fact, in vivo electrophysiological studies have shown that pyramidal projection neurons and interneurons in the basolateral amygdala fire at opposite phases of entorhinal theta (Paré and Gaudreau, 1996).

Although glutamatergic pyramidal cells are the main cell type involved in the interconnections of the various cortical structures of the medial temporal lobe memory system, recent studies suggest that a small number of GABAergic nonpyramidal neurons participating in these interconnections are critical for synchronizing oscillatory activity in these structures (Jinno et al., 2007, Melzer et al., 2012, Caputi et al., 2013). In fact, all major portions of the medial temporal lobe memory system are interconnected by various subpopulations of nonpyramidal GABAergic projection neurons (Jinno, 2009, Caputi et al., 2013). The main amygdalar nuclei projecting to the entorhinal cortex and other cortices of the medial temporal lobe memory system are the basolateral and cortical nuclei (the corticobasolateral nuclear complex of the amygdala; CBL) which contain neurons that resemble those of the cerebral cortex (McDonald, 1992a, McDonald, 2003, Sah et al., 2003). The principal neurons in the CBL are pyramidal-like projection neurons that utilize glutamate as an excitatory neurotransmitter, whereas most nonpyramidal neurons in the CBL are interneurons that utilize GABA as an inhibitory neurotransmitter (McDonald, 1982, McDonald, 1985, McDonald, 1992a, McDonald, 1992b, McDonald, 1996, McDonald, 2003, Millhouse and DeOlmos, 1983, Fuller et al., 1987, Carlsen and Heimer, 1988, McDonald and Augustine, 1993). Like the cortex, the CBL contains at least four distinct subpopulations of GABAergic nonpyramidal neurons that can be distinguished on the basis of their content of calcium-binding proteins and peptides, including: (1) parvalbumin (PV), (2) somatostatin (SOM), (3) vasoactive intestinal peptide (VIP), and (4) cholecystokinin (CCK) (Kemppainen and Pitkänen, 2000, McDonald and Betette, 2001, McDonald and Mascagni, 2001, McDonald and Mascagni, 2002, Mascagni and McDonald, 2003, Mascagni et al., 2009). In addition, it has been demonstrated that the expression of neuropeptide Y (NPY) defines a distinct subpopulation of SOM+ neurons in the CBL (McDonald, 1989, McDonald et al., 1995). A previous study that mainly focused on the interconnections of the parahippocampal region, but also included the lateral amygdalar nucleus in the same horizontal sections, demonstrated that some NPY neurons were involved in the interconnections of these areas, including nonpyramidal NPY+ neurons in the lateral nucleus that had projections to the entorhinal cortex (Köhler et al., 1986). The present study combined Fluorogold (FG) retrograde tract tracing with immunohistochemistry for nonpyramidal cell markers to more thoroughly investigate the involvement of nonpyramidal neurons in the projections of the amygdala to the entorhinal region.

Section snippets

Injections and tissue preparation

A total of 11 adult male Sprague–Dawley rats (250–350 g; Harlan, Indianapolis, IN, USA) received either unilateral (n = 9) or bilateral (n = 2) injections of FG into some of the main cortical areas targeted by the basolateral amygdala (Pitkänen et al., 2002). The bilateral injections (all into the entorhinal/subicular region) were made after an analysis of rats with unilateral injections of this region revealed that the average number of retrogradely labeled neurons in the contralateral amygdala was

Pattern of FG retrograde labeling in the amygdala

Eight rats received injections into the parahippocampal region. Since two of these rats had bilateral injections, and there was no significant contralateral projection (see above), there were a total of 10 cases with these injections. Cases with injections that had extensive involvement of the rostral portion of the dorsolateral entorhinal area (DLEA; R13 and R39) had numerous FG+ retrogradely labeled neurons in most nuclei of the amygdala (Fig. 1, Fig. 10, Fig. 2, Fig. 3). However, FG+ neurons

Pattern of retrograde labeling in the amygdala

The overall pattern of retrograde labeling seen in the amygdala in the present study was consistent with the findings of previous tract tracing studies of amygdalocortical projections (for a review see Pitkanen, 2000). The novel finding of the present study is that a small number of SOM+ nonpyramidal neurons, located mainly in the basolateral and cortical nuclei of the amygdala, take part in amygdalo-entorhinal and amygdalo-parasubicular projections, but not projections to the prefrontal

Acknowledgements

The authors are grateful for the generous donation of the guinea pig FG antibody obtained from Dr. Lothar Jennes (University of Kentucky) and the mouse SOM antibody obtained from Dr. Alison Buchan (University of British Columbia). This work was supported by National Institutes of Health Grant R01-DA027305.

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