Cholinergic interneurons are differentially distributed in the human striatum

Abstract

Background: The striatum (caudate nucleus, CN, and putamen, Put) is a group of subcortical nuclei involved in planning and executing voluntary movements as well as in cognitive processes. Its neuronal composition includes projection neurons, which connect the striatum with other structures, and interneurons, whose main roles are maintaining the striatal organization and the regulation of the projection neurons. The unique electrophysiological and functional properties of the cholinergic interneurons give them a crucial modulating function on the overall striatal response.

Methodology/principle findings: This study was carried out using stereological methods to examine the volume and density (cells/mm(3)) of these interneurons, as visualized by choline acetyltransferase (ChAT) immunoreactivity, in the following territories of the CN and Put of nine normal human brains: 1) precommissural head; 2) postcommissural head; 3) body; 4) gyrus and 5) tail of the CN; 6) precommissural and 7) postcommissural Put. The distribution of ChAT interneurons was analyzed with respect to the topographical, functional and chemical territories of the dorsal striatum. The CN was more densely populated by cholinergic neurons than the Put, and their density increased along the anteroposterior axis of the striatum with the CN body having the highest neuronal density. The associative territory of the dorsal striatum was by far the most densely populated. The striosomes of the CN precommissural head and the postcommissural Put contained the greatest number of ChAT-ir interneurons. The intrastriosomal ChAT-ir neurons were abundant on the periphery of the striosomes throughout the striatum.

Conclusions/significance: All these data reveal that cholinergic interneurons are differentially distributed in the distinct topographical and functional territories of the human dorsal striatum, as well as in its chemical compartments. This heterogeneity may indicate that the posterior aspects of the CN require a special integration of information by interneurons. Interestingly, these striatal regions have been very much left out in functional studies.

Figure 1. Topographical subdivisions of the human caudate nucleus (CN) and putamen (Put) and general pattern of distribution and volume measurements of striatal cholinergic interneurons.

A, Drawing of a sagittal view of the striatum illustrating the various territories of the CN and Put examined in this study. Continuous lines indicate the boundaries between adjacent territories. a–d, Neurolucida drawings illustrating the distribution of the cholinergic neurons in four coronal sections of the striatum depicted in an anteroposterior order. The anteroposterior levels of these sections are indicated by dashed lines in A, and the various sectors in which each striatal territory was subdivided in the present study are depicted by dashed lines in a–d. B–E, photomicrographs showing various cholinergic interneurons with an ovoid (B), globular (C), fusiform (D) and triangular (E) perikaryon. F, bar graph illustrating the mean volume of the cell bodies of the cholinergic cells in the different striatal territories. Significant (*, 0.05>P>0.01) and highly significant (**, P<0.01) differences are indicated. CN, caudate nucleus; n, number of cells analyzed in each striatal region; Put, putamen. Scale bar in B, 40 µm.

Figure 2. Variation in the density of the cholinergic interneurons throughout the striatum.

A, line graph showing the mean density of the cholinergic neurons (cells/mm³) in the various striatal territories of the CN and Put. B–E, bar graphs illustrating the mean density of cholinergic cells in the various sectors (i.e. dm, vm, dl, vl) of the following striatal territories: CN precommissural head (B), CN postcommissural head (C), precommissural Put (D) and postcommissural Put (E). Significant (*, 0.05>P>0.01) and highly significant (**, P<0.01) differences are indicated. dl, dorsolateral; dm, dorsomedial; pv, posteroventral; vl, ventrolateral; vm, ventromedial.

Figure 3. Neuronal density of the cholinergic interneurons in the different functional territories of the human CN and Put.

A, tridimensional schema showing the associative, sensorimotor and limbic territories of the CN and Put , . a–d, drawings illustrating the functional striatal territories in the coronal plane. The anteroposterior level of each coronal drawing is indicated in the tridimensional schema of the striatum. B, table indicating the functional territory represented in each topographical region in which the striatum was subdivided. The functional domains overlap in some sectors of the CN head, in the CN tail and in the precommissural Put. C, bar graph showing the mean density of cholinergic interneurons (mean±SEM) estimated in the associative, sensorimotor and limbic territories of the dorsal striatum. Please note that the data shown in this bar graph were obtained by analyzing only those sectors of the striatum that were exclusively associative, sensorimotor or limbic, as shown in B. D, graph illustrating the cholinergic neuronal density in the various functional territories that are present in every topographical region of the CN and Put. The legend indicates the single or convergent nature of the functional territories. Highly significant (**, P<0.01) differences are indicated in C. CN, caudate nucleus; dl, dorsolateral; dm, dorsomedial; Put, putamen; pv, posteroventral; vl, ventrolateral; vm, ventromedial.

Figure 4. Distribution patterns of the cholinergic interneurons within the striosomes in the various territories of the CN and Put.

A–F, photomicrographs and camera lucida drawings depicting the distribution of the cholinergic interneurons in six enkephalin-immunoreactive striosomes located in the precommissural head (A), postcommissural head (B), body (C) and gyrus (E) of CN, and in the precommissural (D) and postcommissural (F) Put. The light and dark gray shadings in A, D and F indicate the center and periphery of the striosomes, respectively. The inset on the bottom right of each drawing indicates the location of the depicted striosome. Scale bar, 650 µm (A), 1000 µm (B), 260 µm (C), 1500 µm (D), 700 µm (E), 1000 µm (F).

Figure 5. Distribution of ChAT-ir interneurons in the striatum and in its chemical compartments.

A, gradients of density of cholinergic interneurons in the various territories of the CN and Put. Within each territory, the density of cholinergic neurons is higher in the quadrants indicated by the asterisks. B, variation in the distribution of the cholinergic interneurons within the striosomes in the various striatal territories. The bar graph illustrates the percentage of striosomes that fulfil the condition indicated by each color in the legend. ENK+ region comprises homogeneous striosomes and the periphery of heterogeneous striosomes. n in the X-axis indicates the number of striosomes (homogeneous/heterogeneous) analyzed in each territory. C, color photomicrograph taken from a double ENK/ChAT immunostained section showing one striosome and numerous ChAT-ir neurons in the precommissural head of CN. Observe that the periphery of the striosome is more intensely stained for ENK than its center, and that the ChAT-ir neurons occur within the striosome and in the surrounding matrix. CN, caudate nucleus; ENK, enkephalin; Put, putamen. Scale bar, 250 µm (C).

Figure 6. Schematic drawings with functional considerations on the ChAT-ir interneurons.

A, difference in the density of cholinergic interneurons between the CN and Put. ChAT-ir neurons populate the matrix and the striosomes in the CN and Put but the number of these cells in the two compartments is higher in the former than in the latter. In both striatal components, the cholinergic interneurons abound at the boundaries between the two major compartments and the two striosomal regions. B, Variation in the activity of the cholinergic neurons defined as TANs depending on: 1, the presence or absence of a reward-associated stimulus and 2, their location in the matrix or within a striosome. a–a′, without stimulus the TANs constantly release ACh, which exerts a dual control on GABAergic interneurons through the nicotinic and muscarinic receptors. Due to the large amount of ACh release by these cholinergic neurons, the greater or lower amount of AChE might not be a limiting factor for the activation of the GABAergic interneuron cholinergic receptors. b–c, in the presence of a stimulus the amount of ACh released by the TANs decreases. If ACh is released in the matrix, the high AChE content of this milieu might stop the ACh signal very rapidly at the synaptic cleft, thereby avoiding nicotinic receptor activation on the GABAergic interneurons, and, therefore, facilitating the discharge of projection neurons. If the ACh release is inside a striosome, its low AChE content could facilitate activation of the nicotinic and muscarinic receptors on the GABAergic neurons, which, in turn, would inhibit the projection neurons. ACh, acetylcholine; AChE, acetylcholinesterase; ChAT-IN, cholinergic interneuron; GABA-IN, GABAergic interneuron; GP, globus pallidus; M, muscarinic receptor; N, nicotinic receptor; PN, projection neuron; SNr, substantia nigra pars reticulata.