Elsevier

Brain Research

Volume 929, Issue 2, 8 March 2002, Pages 243-251
Brain Research

Research report
Calbindin D-28k positive projection neurones and calretinin positive interneurones of the rat globus pallidus

https://doi.org/10.1016/S0006-8993(01)03263-2Get rights and content

Abstract

Immunohistochemistry for three calcium-binding proteins calbindin D-28k, calretinin, and parvalbumin revealed neuronal heterogeneity within the GP. Each neurone appeared to express either a single type of calcium binding protein or none at all. The co-localisation of calcium binding proteins was not observed. Combined immunohistochemistry and retrograde tract tracing using colloidal gold particles injected into the projection fields, the substantia nigra or subthalamic nucleus, revealed that projection neurones could be labelled with either calbindin or parvalbumin. These cells were of medium size (22×12 μm), multipolar and moderate varicose dendritic trees. In contrast, calretinin-positive neurones were never retrogradely labelled, even in regions where neuronal colloidal gold deposits were numerous. This, combined with their rarity (<1%) and small size (11×9 μm), suggests that calretinin may be a neurochemical marker for putative rat globus pallidus interneurones. Calcium-binding proteins are known to have unique buffering characteristics that may confer specific functional properties upon pallidal neurones. Indeed, differential calcium binding protein expression may underlie the electrophysiological heterogeneity observed in the rat globus pallidus.

Introduction

Until recently, the globus pallidus (GP: equivalent to the external segment of the primate globus pallidus) was viewed as a simple structure composed of a homogenous population of neurones which act as relays between the striatum and the subthalamic nucleus. With advances in techniques, this view has recently been challenged and the GP is now considered a heterogeneous structure which has a wide range of connections throughout the basal ganglia [41] including the internal segment of the globus pallidus [21] and the substantia nigra pars reticulata [40], the striatum [42], the reticular thalamic nucleus [39] and the pedunculopontine region [28].

Morphological studies using the Golgi technique [25], [17] and intracellular staining combined with electrophysiology [33], [24] have revealed multiple GP neuronal subtypes. In vivo, multiple subtypes of rat GP neurone have been identified on the basis of firing pattern and waveform [23], while sharp microelectrode studies in vitro have indicated that the guinea pig GP contains three neuronal subtypes [29], [30]. Our own patch clamp studies in slices of rat GP have identified two major populations of GP neurones which can be distinguished on the basis of membrane properties and morphology [45].

Differences in neuronal electrophysiological characteristics are often accompanied by differential neurochemical expression. At the macroscopic level, the GP exhibits a complementary pattern of parvalbumin (PV) and calbindin (CB) neuropil expression [10], [36]. However, to date, only PV positive cell bodies have been observed in the rat GP [15], [36] although CB positive [10], [32] and calretinin (CR) positive neurones [13], [32] have been observed in the GPe of the primate.

The aim of the current study was to determine whether pallidal neurones could be identified on the basis of somal PV, CB or CR expression and whether there is evidence for the co-localisation CaBPs as in other areas of the basal ganglia such as substantia nigra pars compacta [38] and subthalamic nucleus [1]. Immunohistochemistry for CaBPs was coupled with retrograde tract tracing to determine whether their differential expression could be used as a marker neuronal phenotype in the GP.

Section snippets

Subjects

Male Sprague–Dawley rats (110–140 g) were used throughout the study in order to permit comparisons with the results of in vitro electrophysiological studies being conducted in the laboratory [45]. The animals were group housed on a 12 h-light/dark cycle and given free access to food and water. All procedures were conducted in accordance with the Animals (Scientific Procedures) Act, 1986, UK.

Retrograde neuroanatomical labelling of globus pallidus neurones

A colloidal gold tracer was stereotaxically injected into 18 animals, which had been anaesthetised with

Distribution of calcium binding protein immunoreactivity

At a macroscopic level, CB positive neuropil staining was highest in the medial parts of the GP, although a thin band of staining was also seen adjacent to the striatal border. CB positive cell bodies (8.35±0.74 cell bodies per GP section; mean±S.E.M.) were observed throughout the GP (Fig. 1A). Cell bodies were multipolar, bipolar or fusiform in shape (mean dimensions 19.92±0.55×11.8±0.24 μm) with smooth or moderately varicose processes (Fig. 2A). Many of the bipolar neurones lay parallel to

Discussion

The results demonstrate that both CB and CR immunopositive neurones were distributed throughout the GP. In keeping with previous studies, many PV positive cell bodies were observed the density of which was greatest in the lateral parts of the structure [4], [15], [22], [36]. Sequential double immunohistochemical processing suggests that these CaBPs be uniquely expressed with no more than one CaBP being localised in a single cell. Furthermore, combined immunohistochemistry and retrograde

Acknowledgements

The authors wish to thank Dr I.J. Mitchell for use of the photomicroscope system and for invaluable assistance in criticising the manuscript. This work was supported by the Wellcome Trust, UK (Grant No. 050196/Z/97/Z) and a Research Project Grant from the Faculty of Medicine and Dentistry, University of Birmingham.

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