Elsevier

Neuroscience

Volume 241, 25 June 2013, Pages 215-228
Neuroscience

Region-specific diversity of striosomes in the mouse striatum revealed by the differential immunoreactivities for mu-opioid receptor, substance P, and enkephalin

https://doi.org/10.1016/j.neuroscience.2013.03.012Get rights and content

Highlights

  • Diversity of striosomes in mouse striatum was studied by immunohistochemistry.

  • Mu-opioid receptor-rich striosomes were located only in rostral half of the striatum.

  • Substance P-positive striosomes were located in rostral two-thirds of the striatum.

  • Labeling for enkephalin and SP showed dorsoventral differences in rostral striosomes.

  • The results suggest site-dependent regulation of striosomal neurons by opioids and SP.

Abstract

The complexity of the internal structure of the striatum is not completely understood, and the striosomes/matrix compartmentalization in particular has been one of the intriguing substructures of the striatum. Although various neurochemical markers have been used to visualize striosomes with sufficient clarity, it still remains obscure whether striosomes that are detectable by a single marker represent all of the striosomal compartments and to what extent the compartments are uniform across different intrastriatal positions. Triple immunohistochemical labeling for the three representative striosomes/matrix markers, μ-opioid receptor (MOR), substance P (SP), and enkephalin (Enk), was applied to serial sections covering the whole striatum of the mouse (n = 8). The majority of MOR-positive striosomes were confined to the rostral quarter of the striatum. In contrast, SP-positive striosomes were distributed more broadly in the rostral two-thirds of the striatum. No striosomes were observable in the caudal third by the present method. In the rostral striatum, the majority of striosomes were labeled for both MOR and SP, but some at the most rostral positions were detectable only by MOR, while caudally located striosomes were identifiable only by SP. Thus MOR- and SP-immunoreactivities in striosomes exhibited contrasting patterns along the rostrocaudal axis. The Enk immunohistochemistry produced complicated profiles and was unsuitable for the detection of striosomes in mice. However, Enk immunoreactivity in MOR and/or SP-positive striosomes was higher in the ventral portion than in the dorsal portion in the rostral striatum. The present study revealed the region-specific diversity of striosomes, suggesting site-dependent differential regulation of striosomal neurons by MOR ligands and SP that are contained in indirect- and direct-pathway neurons, respectively. The results further suggest the necessity of viewing the striosomes as non-uniform compartments in addition to the traditional dichotomous view, which focuses on discrimination between the striosomes and the matrix.

Introduction

The striatum forms the major input structure of the basal ganglia. It receives driving inputs mainly from the cerebral cortex, sends efferent axons toward other components of the basal ganglia, and is involved in the control of a wide variety of behaviors such as motor activities, cognition, working memory, and reward-related action (Pennartz et al., 2009, Crittenden and Graybiel, 2011).

One important feature in the internal structure of the striatum is that axons from individual cortical areas form longitudinal parallel circuits that are arranged in a mosaic-like fashion when viewed in a coronal plane (Selemon and Goldman-Rakic, 1985, Malach and Graybiel, 1986, Brown et al., 1998). These mosaic-like parallel circuits are segregated from each other, but some form of interaction has also been demonstrated between them, suggesting the associative function of the striatum (Malach and Graybiel, 1986).

The striatal mosaic is also represented as the two distinct compartments inside the striatum, striosomes (patches) and matrix. The striosomes were first recognized as compartments of low acetylcholine esterase activity in the striatum of the cat, monkey and human (Graybiel and Ragsdale, 1978). Since then, the striosomes have been visualized in many species by using numerous chemical markers (Graybiel et al., 1981a, Graybiel et al., 1981b, Beach and McGeer, 1984, Gerfen et al., 1985, Holt et al., 1997, Prensa et al., 1999; for a comprehensive list, see Crittenden and Graybiel, 2011). Many studies on the comparison between striosomes and matrix have added essential information on how the basal ganglia promote appropriate behaviors, and they also provide a novel viewpoint to understand several neurological disorders, such as Parkinson’s disease, Huntington’s disease, dystonia, and drug addiction (Crittenden and Graybiel, 2011).

Various staining techniques have been used to visualize the striatal compartmentalization with sufficient clarity. However, it remains unclear to what extent all of the striosomal or matrix regions belong to the uniform compartment. In fact, the comparison of the immunostaining among various striosomal markers has already revealed complicated labeling patterns; some striosomes are intensely labeled by both of two different markers such as substance P (SP) and enkephalin (Enk), whereas others by only either of the two (Graybiel et al., 1981b, Martin et al., 1991, Prensa et al., 1999). This pattern raises the possibility that individual chemical markers that clearly demarcate striosomes do not necessarily cover all of the striosomes when used alone. Furthermore, topographical differences in the labeling of striosomal markers have been noted by the analysis in the whole extension of the striatum in the cat (Graybiel et al., 1981b), monkey (Martin et al., 1991) and human (Holt et al., 1997, Prensa et al., 1999, Bernácer et al., 2008). In this respect, the data on the anatomy of the compartmentalization in mice striatum are rather limited. Especially the detectability of striosomes across the entire striatum has not been addressed so far in mice. Mice are the most frequently used animals currently in gene-targeted experiments, and increasing numbers of functional studies have been performed in mice to reveal the underlying mechanisms of striatal control of behaviors under both physiological and pathological conditions. It is, therefore, valuable to have a clear understanding of the anatomy of the striosomes/matrix organization in mice.

In this study, we explored the diversity of the mice’s striosomes by examining the immunohistochemical staining patterns for the four representative substances visualizing the striosomes/matrix (mu-opioid receptor, MOR; SP; Enk; calbindin), and three of them (MOR, SP, Enk) were analyzed quantitatively in coronal sections. The immunoreactivities for these were compared directly in triple-immunolabeled specimens using confocal laser scanning microscopy (CLSM), surveyed throughout the rostrocaudal extent of the striatum as well as along its dorsoventral axis. The obtained results suggest the necessity for taking the diversity of striosomes and its site-dependency into consideration in the analysis of the striatal circuitry and function, such as exploration of the possible afferent connectivity between the limbic cortices and striosomes and the efferent connectivity from the striosomes to dopaminergic neurons in the substantia nigra pars compacta.

Section snippets

Tissue preparation

All of the experiments and animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publications No. 80-23, revised 1996), and all of the protocols were approved by the Institutional Animal Care and Use Committee at the Kyushu University. All of the efforts were made to minimize the number of animals used and their suffering.

Eight male C57BL/6J mice (25–30 g, 6–8 weeks old) were deeply anesthetized with sodium

MOR- and SP-positive striosomes

The rostrocaudal extent of the striatum was assessed by observations of coronal sections that were cut serially and processed for Nissl staining. The striatum was situated between 1600 μm rostral to the anterior commissure (AC; +1600 μm) and 2200 μm caudal to AC (−2200 μm). We first labeled serial sections with different antibodies and sought which immunohistochemical marker most broadly visualizes the compartments along this rostrocaudal extent of the striatum. Triple labeling using antibodies

Discussion

The present study has revealed the site-dependent diversity of mice striosomes, which was recognizable as non-uniform staining patterns along both the rostrocaudal and dorsoventral axes of the striatum. The main results are summarized as follows:

  • (1)

    MOR and SP provide the best labeling of striosomes in mice striatum.

  • (2)

    MOR-positive striosomes are located in the rostral half of the striatum, with the majority confined to the rostral quarter.

  • (3)

    SP-positive striosomes are located in the rostral two-thirds

Acknowledgements

The authors thank Sawai K and Tanaka C for excellent technical assistance and Dr. T. Kosaka for carefully reading the manuscript and providing useful comments. This work was supported by Grants-in-Aid for Science Research from the Japan Society for the Promotion of Science (18300108, 21500327, 24300127).

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