Research reportDifferences in the laminar origin of projections from the medial prefrontal cortex to the nucleus accumbens shell and core regions in the rat
Introduction
The prefrontal cortex (PFC) in mammals is commonly defined as the ‘essential cortical projection area of the mediodorsal nucleus of the thalamus’ [22], [34], [48], [51]. It is composed of several cortical sub-regions, reciprocally connected with parts of the mediodorsal nucleus, and is divided into medial and lateral divisions [22], [34]. In the rat the medial prefrontal cortex (mPFC), is an agranular region of the cortex located over the rostral cerebral hemispheres [34], [47]. It consists of the medial precentral area, dorsal anterior cingulate cortex, prelimbic and infralimbic cortices and is divided into distinct cytoarchitectonic layers (I–VIb) with no layer IV [7], [18], [34]. Within the mPFC exist different populations of local circuit, GABAergic interneurons (15% of total neurons) and pyramidal projection neurons (85%) [15], [16], [17], [18].
In humans the different sub-regions of mPFC are thought to be involved in a variety of activities, including control of autonomic functions (infralimbic and ventral portions of prelimbic) [24], [27], [40] and motivation, cognition, spatial tasks and blood pressure control [32], [33], [46], [54], [55].
It has long been thought that some of the functions involving the mPFC are due, in part, to the neuronal connections it establishes with the dorsal and ventral striatum. The ventral striatum or nucleus accumbens is that part of the striatal complex proposed to be most directly involved with the limbic system [19], [21]. Based on the distribution patterns for various neuroactive substances and receptors [6], [38], [56], [62], [63], synaptic organisation [39], [61], dopamine metabolism [9], cytoarchitecture [37] and electrophysiological response properties [41], [42] the nucleus accumbens is considered to consist of three main sub-divisions. These include a central ‘core’ which ensheathes the anterior limb of the anterior commissure and gradually merges into the ventral part of the dorsal striatum, a ‘shell’ which comprises medial, ventral and lateral divisions that surround the core and finally a ‘rostral pole’ where core and shell regions cannot easily be differentiated.
Previously, the striatal projections of PFC neurons have been examined using both anterograde and retrograde tracing techniques [2], [4], [14], [20], [36], [49]. From these investigations it has been shown that PFC neurons target the striatum in a topographical fashion, and that areal and laminar origin of cortical projection fibres maybe related to a compartmental distribution of terminals within the striatum [2], [4], [49]. Nevertheless, the precise details of how this might be organised are still unclear. In this study we set out to investigate the morphological identity, laminar distribution and precise cytoarchitecture of prefrontal neurons that project to nucleus accumbens shell and core compartments.
Neurobiotin (N-2-aminoethyl biotinamide hydrochloride) was originally introduced for the purpose of intracellular neuron filling following electrophysiological recordings [25], [64]. However it has since been shown to be a robust and consistent neuronal tract tracer even when injected iontophoretically [10], [11], [26], [35], largely due to its small molecular weight, large solubility, high rate of transport (1–2 mm/h) [29], and ease of visualisation [26], [35], [52].
The major aims of this study were (a) to exploit the advantages of Neurobiotin as a neuronal tract-tracer to investigate the morphology of mPFC neurons that target the nucleus accumbens and (b) to compare the laminar distributions of those neurons that innervate the shell and core regions of the nucleus accumbens.
Section snippets
Animals and tissue preparation
All experiments were performed in accordance with the U.K. Animals (Scientific Procedures) Act of 1986. Six male Wistar rats (200–250 g) were used in this study. Subjects were deeply anaesthetised (intra-peritoneal) with a Hypnorm/Hypnovel mixture (0.27 ml/100 g) [59] and mounted into a stereotaxic frame. Neurobiotin [6% in 0.05 M Tri-sodium-buffered saline (TBS), Vector Labs] was delivered iontophoretically (7 s at 7 μA and 5 s off, using a positive/cathodal current for 10–20 min), into
Results
The injected Neurobiotin was visualised by the peroxidase reaction product formed using ABC reagent. Labelled structures contained a black (Ni–DAB) or grey (SG) insoluble precipitate, and were homogeneously labelled. Neurobiotin was apparently taken up by axon terminals at the injection site and transported in a retrograde fashion back to the cells of origin. In control sections, incubated without the ABC reagent, no specific histochemical staining was observed.
Discussion
This study is the first to demonstrate that Neurobiotin not only identifies the location of neurons in the mPFC that project to the nucleus accumbens, but also reveals the morphology of these projection neurons in exquisite detail. Furthermore, it shows that populations of pyramidal projection neurons in deep layers V and VI throughout the rostro-caudal extent of prelimbic and infralimbic cortex innervate both core and shell regions of the nucleus accumbens. Meanwhile neurons in superficial
Acknowledgements
The comments and advice of Professor J.P. Bolam are greatly appreciated. This work is supported by a British Medical Research Council Studentship (D.C.D. Ding).
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