The Stereological Analysis and Spatial Distribution of Neurons in the Human Subthalamic Nucleus

Abstract

The subthalamic nucleus (STN) is a small, ovoid structure, and an important site of deep brain stimulation (DBS) for the treatment of Parkinson’s disease. Although the STN is a clinically important structure, there are many unresolved issues with regard to it. These issues are especially related to the anatomical subdivision, neuronal phenotype, neuronal composition, and spatial distribution. In this study, we have examined the expression pattern of 8 neuronal markers [nNOS, NeuN, parvalbumin (PV), calbindin (CB), calretinin (CR), FOXP2, NKX2.1, and PAX6] in the adult human STN. All of the examined markers, except CB, were present in the STN. To determine the neuronal density, we have performed stereological analysis on Nissl-stained and immunohistochemical slides of positive markers. The stereology data were also used to develop a three-dimensional map of the spatial distribution of neurons within the STN. The nNOS population exhibited the largest neuronal density. The estimated total number of nNOS STN neurons is 281,308 ± 38,967 (± 13.85%). The STN neuronal subpopulations can be divided into two groups: one with a neuronal density of approximately 3,300 neurons/mm3 and the other with a neuronal density of approximately 2,200 neurons/mm3. The largest density of STN neurons was observed along the ventromedial border of the STN and the density gradually decreased toward the dorsolateral border. In this study, we have demonstrated the presence of 7 neuronal markers in the STN, three of which were not previously described in the human STN. The human STN is a collection of diverse, intermixed neuronal subpopulations, and our data, as far as the cytoarchitectonics is concerned, did not support the tripartite STN subdivision.

Transcriptional Profile of the Developing Subthalamic Nucleus

“…The literature search for studies of human STN revealed there are hardly any studies that analyzed the molecular phenotype or developmental origin of STN neurons. The majority of studies dealing with human STN analyzed expression patterns of calcium-binding proteins and neurotransmitter receptors in STN ( Lévesque and Parent, 2005 ; Zwirner et al, 2017 ; Wu et al, 2018 ; Alkemade et al, 2019 ), with only one study also analyzing TF expression ( Bokulić et al, 2021 ). However, all these studies analyzed the adult STN, so the data about TFs in the developing human STN is lacking.…”

“…In mice, Nkx2-1 has now been acknowledged as a marker of pallidal neurons originating from the MGE, expressing parvalbumin, projecting to the STN, and having a high-frequency firing rate ( Abdi et al, 2015 ; Dodson et al, 2015 ). Interestingly, a recent study of the adult human STN described the expression of NKX2-1 in the STN, along with parvalbumin and calretinin ( Bokulić et al, 2021 ). As mentioned before, postmitotic neurons destined to populate the mouse STN downregulate Nkx2-1 , so the observed difference between human and mouse STN could indicate the existence of an additional neuronal subclass or a species-specific mechanism regulating its expression.…”

“…Interestingly, adult mouse STN apparently has no Foxp1 mRNA signal ( Tamura et al, 2004 ; Philips et al, 2005 ). In the developing human brain, both FOXP1 and FOXP2 were expressed in the STN during midgestation (22 postconceptional weeks); however, FOXP2 expression was stronger ( Teramitsu et al, 2004 ), and the FOXP2 protein can be detected in adult STN ( Bokulić et al, 2021 ). These postmitotic markers were found in human fetal striatum at 11 gestational weeks, so perhaps they are also expressed in the STN earlier than previously reported ( Pauly et al, 2013 ).…”

“…Also, Nkx2-1 is clearly indispensable for the formation of the basal hypothalamus in rodent models, yet it is absent from the STN from E12.5 onward ( Kimura et al, 1996 ; Nakamura et al, 2001 ; Kim et al, 2020 ; Murcia-Ramón et al, 2020 ). Nevertheless, there have been reports of NKX2-1 expression in the adult human STN ( Bokulić et al, 2021 ), so these discrepancies between model animals and humans remain to be further investigated.…”

“…However, clinical and preclinical neuroimaging studies have not studied intra-basal ganglia connectivity or possible abnormalities of small nuclei like STN in RRBs (for review, see Wilkes and Lewis, 2018 ), although animal models demonstrated clear involvement of STN in stereotyped behavior ( Chang et al, 2016 ; Lewis et al, 2018 ; Wilkes and Lewis, 2018 ). Genes implicated in speech impairment ( FOXP1 and FOXP2 ) have widespread expression in the basal ganglia, STN included ( Enard, 2011 ; Bacon and Rappold, 2012 ; Co et al, 2020 ; Bokulić et al, 2021 ). These expression patterns, coupled with the role of basal ganglia in language acquisition ( Eigsti et al, 2011 ; Enard, 2011 ) and speech impairment being recognized as a side effect of DBS ( Pützer et al, 2008 ; Silveri et al, 2012 ; Ehlen et al, 2014 ), point to a possible involvement of STN in speech production.…”