DARPP-32 Cells and Neuropil Define Striosomal System and Isolated Matrix Cells in Human Striatum

CJ Arasaratnam; JJ Song; T Yoshida; MA Curtis; AM Graybiel; RLM Faull; HJ Waldvogel

doi:10.1002/cne.25473

. Author manuscript; available in PMC: 2024 Jun 1.

Published in final edited form as: J Comp Neurol. 2023 Mar 31;531(8):888–920. doi: 10.1002/cne.25473

DARPP-32 Cells and Neuropil Define Striosomal System and Isolated Matrix Cells in Human Striatum

CJ Arasaratnam ¹, JJ Song ¹, T Yoshida ², MA Curtis ¹, AM Graybiel ², RLM Faull ¹, HJ Waldvogel ¹

PMCID: PMC10392785 NIHMSID: NIHMS1880284 PMID: 37002560

Abstract

The dorsal striatum forms a central node of the basal ganglia interconnecting the neocortex and thalamus with circuits modulating mood and movement. Striatal projection neurons (SPNs) include relatively homogeneously intermixed populations expressing D1-type or D2-type dopamine receptors (dSPNs and iSPNs) that give rise to the direct (D1) and indirect (D2) output systems of the basal ganglia. Overlaid on this D1–D2 organization is a macroscopic compartmental organization, in which a labyrinthine system of interconnected striosomes made up of sequestered SPNs is embedded within the much larger striatal matrix. Striosomal SPNs also include D1-SPNs and D2-SPNs, but they can as well be distinguished from matrix SPNs by many neurochemical and gene-expression markers. In the well-studied striatum of rodents, the key signalling molecule, DARPP-32, is a conspicuous exception to these generally compartmental expression patterns, thought to befit its widespread functions through opposite actions in both D1- and D2-expressing SPNs. We demonstrate here, however, that in the dorsal human striatum, DARPP-32 is highly concentrated in the neuropil and SPNs of striosomes, especially in the caudate nucleus and dorsomedial putamen, relative to the neuropil of the matrix in these regions. The generally DARPP-32-poor matrix contains widely scattered DARPP-32-positive cells. DARPP-32 cell bodies in both compartments proved negative for conventional intraneuronal markers examined. These findings raise the potential for specialized DARPP-32 expression in the human striosomal system and in a set of otherwise unidentified DARPP-32-positive neurons in the matrix. If this DARPP-32 immunohistochemical positivity predicts differential functional DARPP-32 activity, then the distributions we demonstrate here could render striosomes and sets of dispersed matrix cells susceptible to differential signalling through cAMP and other intracellular signalling systems in health and disease.

Keywords: DARPP-32, Striatum, Striosomes, Spiny Projection Neurons

Graphical Abstract

graphic file with name nihms-1880284-f0001.jpg

The dorsal striatum forms a central node of the basal ganglia interconnecting the neocortex and thalamus with circuits modulating mood and movement. A large majority of the cells within the striatum are the striatal projection neurons (SPNs). Overlaid on this network of SPNs is a macroscopic compartmental organization i.e. the striosomal network, which is embedded within the much larger striatal matrix. Within the wider literature, two SPN markers have been heavily relied upon previously. In the human, the SPN marker of choice has been calbindin, while in the rodent, the SPN marker of choice has usually been DARPP-32. In this article, we demonstrate with qualitative and quantitative methods that in the dorsal human striatum, DARPP-32 is highly concentrated in the neuropil and SPNs of striosomes, especially in the caudate nucleus relative to the neuropil of the matrix, and the generally DARPP-32-poor matrix contains widely scattered DARPP-32-positive cells. However, in the rat the immunohistochemical distribution of DARPP-32 in SPNs or SPN-like cells seems to be relatively similar across both the striosomal and matrix compartments, regardless of whether these cells are colocalised with calbindin or not.

1. INTRODUCTION

The striatum forms a central input-output station of the basal ganglia. Its main neurons are the spiny projection neurons (SPNs), which receive massive inputs from the neocortex and thalamus and project to the output stations of the basal ganglia (Parent, 1990; Parent & Hazrati, 1995). These SPNs can be divided by their expression of either D1 or D2 dopamine receptors, and they give rise to the direct (D1) and indirect (D2) output pathways of the basal ganglia, thought to act in context-dependent opposition to each other or in synergistic patterns to modulate motor control (Bateup et al., 2010; Hersch et al., 1995; Surmeier, Ding, Day, Wang, & Shen, 2007). The D1-dSPNs (dopamine receptor 1 positive direct spiny projection neurons) and D2-iSPNs (dopamine receptor 2 positive indirect spiny projection neurons) are intermixed and apparently homogeneously distributed throughout the striatum, but an orthogonal neurochemical compartmentalization also is present, whereby the striatum is divided into molecularly distinct striosomes and matrix (Graybiel & Ragsdale, 1978; Groves, Martone, Young, & Armstrong, 1988; Tippett et al., 2007). The matrix is large, contains most of the SPNs giving rise to the direct and indirect pathways, and therefore is thought to be in charge of the coordinated modulation of movement by the striatum. The striosomes, which form a three-dimensionally extended labyrinthine mazework within the matrix, are thought to be more related to the limbic system or, more generally, to the selection process imposed on action control by striatal activity. Anatomically, the striosomal labyrinth in sections through the striatum appears as widely distributed clustered zones, about 0.5–1 mm in diameter in the human striatum. Most molecular expression patterns differ for the striosomes and the large matrix around them (Gerfen, Baimbridge, & Miller, 1985; Graybiel & Ragsdale, 1978; Holt, Graybiel, & Saper, 1997; Morigaki & Goto, 2016; Waldvogel & Faull, 1993; H. Waldvogel, Kubota, Fritschy, Mohler, & Faull, 1999). Experimental studies suggest that parts of the striosomal system project directly to the dopamine-containing cells of the substantia nigra—unlike most SPNs of the matrix (Crittenden et al., 2016; Fujiyama et al., 2011); and in vitro slice experiments suggest that they can block spike activity of the dopaminergic cells, with a following rebound activation (Evans et al., 2020; McGregor et al., 2019). Studies of striosomes in rodents and monkeys suggest that anteriorly placed striosomes function in relation to value evaluation, related to affective state (Amemori, Graybiel, & Amemori, 2021). Parts of the striosomal system, especially in anterior striatum, thought to correspond in part to the caudate nucleus in the human, appear to have special connectivity with limbic system circuitry and have been found to affect learning and cost-benefit decision-making in motivationally challenging contexts (Crittenden & Graybiel, 2011; Friedman et al., 2020). Much of the information so far established about these two apparently orthogonal organizations has come from work on rodents, but studies are adding to this body of evidence in non-human primates and implementation of single-nucleus RNA sequencing (snRNA-seq) and related methods for whole genome analysis have opened up the possibility of deep analysis of the cellular identities in humans (He et al., 2021; Märtin et al., 2019; Matsushima et al., 2023).

Despite well-developed immunohistochemical methods of imaging striosomes in post-mortem human brain tissue, it is only in recent years that the prospect of in vivo imaging of putative striosomal connections has become possible (Waugh et al., 2022). Striosomes themselves remain beyond the resolution of in vivo fMRI imaging. There is thus limited evidence for the precise functions of striosomes in human. It nevertheless has been proposed that striosomes aid in exercising global dopamine transmission on basal ganglia circuitry and in maintaining limbic circuitry (Crittenden & Graybiel, 2011; Tippett et al., 2007). Evidence from some post-mortem brain studies have suggested their differential vulnerability in disease states, most notably in Huntington’s disease (Tippett et al., 2007); and this evidence has been confirmed by snRNA-seq analysis (Lee et al., 2020; Matsushima et al., 2023). Still needed, however, are better methods for anatomical work allowing reliable and robust labelling of these systems in post-mortem human brains, including prized biological material for analysing striatal organization in the wake of disease.

To meet this need, we here used immunohistochemical methods in a search for the distribution of DARPP-32, a key second messenger critical to the functional actions of dopamine in the striatum (Greengard, Allen, & Nairn, 1999; Ouimet & Greengard, 1990; Ouimet, Miller, Hemmings, Walaas, & Greengard, 1984). DARPP-32 has been shown to be expressed in both D1 and D2 SPNs, and to act oppositely in them (Bateup et al., 2010), exciting cAMP-related signalling through D1 receptors and inhibiting cAMP-related signalling through D2 receptors. Almost nothing is yet known about potential differential regulation by DARPP-32 in striosomes and matrix. Most anatomical studies have suggested that DARPP-32, like D1 and D2 SPNs, is not particularly enriched in one or the other compartment, but these studies have largely been done for rodents.

Previous studies have indicated that the distribution of DARPP-32 appears to be fairly similar between species such as rats, monkeys and humans, and that DARPP-32 is highly concentrated in dopamine-recipient neurons (Svenningsson et al., 2004). Svenningsson et al. (2004) state that the representation of DARPP-32 in humans is synonymous to DARPP-32 representation in rat striatum. However, few studies to date have used DARPP-32 as a SPN marker in post-mortem human brain tissue (Guo et al., 2012; Hayakawa et al., 2013; Morigaki & Goto, 2016; Straccia, Carrere, Rosser, & Canals, 2016). Traditionally, it is reported that calbindin identifies the majority of SPNs (Ferrante, Kowall, & Richardson, 1991). However, calbindin representation in the striatum is not limited to a marker of SPNs, and calbindin can also be used for consideration of the striosome and matrix network anatomy because it has low expression in striosomes and also in the dorsolateral part of the rodent caudoputamen, in addition to other markers. The original work identifying striosomes was by use of acetylcholinesterase staining in human and non-human primate showed the striosomes as distinct, ~1 mm diameter zones of pale staining embedded in a background of more intense acetylcholinesterase stain (matrix) (Graybiel & Ragsdale, 1978). Later studies have demonstrated that many anatomically detectable molecular species have differential striosome-matrix distributions either during development or at maturity or both, with some significant species differences and regional gradients (Burke & Baimbridge, 1993; Crittenden & Graybiel, 2011; Ferrante et al., 1991; Tippett et al., 2007). Calbindin has been used as a negative marker for more medial parts of the striosomal system, and as a positive matrix marker, across a number of studies (Ferrante et al., 1991; Kuo & Liu, 2020; Tippett et al., 2007).

Direct comparisons between DARPP-32 and calbindin as a cellular marker, and as a negative marker of striosomes, have not so far been available. Here we present such a comparison, wherein we asked whether we could achieve strong immunostaining for DARPP-32 in sections from post-mortem human brain specimens, and whether, if so, we could map its distribution. We found that immunohistochemically detectable DARPP-32, as seen with 3 different antibodies, was strikingly enriched in the neuropil and cells of striosomes, relative to those of the surrounding matrix, and that this pattern was especially vivid in the caudate nucleus and adjoining dorsomedial putamen. We identified, in addition, isolated, widely distributed, intensely immunostained DARPP-32 cells in the otherwise DARPP-32-poor matrix. The findings in post-mortem human striatum were assessed alongside re-characterization of DARPP-32 expression in post-mortem rat tissue, to provide a species comparison. Our findings offer, we suggest, not only a practical benefit in the clarity of marking of the human striosomal system, but also a base for work to identify both molecular and physiological attributes of the human striatum and its striosome and matrix compartments.

2. MATERIALS AND METHODS

2.1. Brain tissue

Human brain tissue was obtained from the Neurological Foundation Human Brain Bank in the Centre for Brain Research at the Faculty of Medical and Health Sciences, University of Auckland, New Zealand. All protocols followed for this work were approved by the University of Auckland Human Participants Ethics Committee, and full consent was received from all the families involved in the process of tissue donation. Brain tissue was obtained from 15 neurologically normal cases, with an average age of 63.2 years (range 41–77 years, post-mortem delay range 8–32 hours) (Table 1) with no apparent display of abnormal neuropathology and no history of neurological disease.

Table 1:

Post-mortem human brain cases used in this study.

	Age (yrs)	Sex	PM delay (hr)	Weight (g)	Cause of death	Procedures
H186	68	M	21	1327	Ischaemic heart disease	Immunohistochemistry (IHC) + cell density measurements + fluoro cell counts
H211	41	M	9.5	1513	Ischaemic heart disease	IHC
H204	66	M	9	1461	Ischaemic heart disease	IHC
H215	67	F	23.5	1239	Ischaemic heart disease	IHC
H231	65	M	8	1527	Ischaemic heart disease	IHC
H160	77	M	23	1490	Ischaemic heart disease	IHC+ cell density measurements + fluoro cell counts
H170	60	M	17	1370	Ischaemic heart disease	IHC + cell density measurements
H230	57	F	32	1243	Carcinoma	IHC
H242	61	M	19.5	1466	Coronary atherosclerosis	IHC + cell density measurements + fluoro cell counts
H243	77	F	13	1184	Ischaemic heart disease	IHC
H146	61	M	15	1488	Ischaemic heart disease	Western blotting
H194	68	M	22.5	1403	Coronary atherosclerosis	Western blotting
H200	56	M	23	1358	Asphyxia	Western blotting + immunoprecipitation
H231	65	M	8	1527	Ischaemic heart disease	IHC
H239	64	M	15.5	1529	Ischaemic heart disease	Western blotting + immunoprecipitation
H241	76	F	12	1094	Metastatic cancer	IHC + Western blotting + fluoro cell counts
H140	51	M	18	-	Dilated cardiomyopathy	Cell density measurements

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Brains received by the Brain Bank were treated as described in Waldvogel et al. (2008). The brains were first weighed, and the brainstem and cerebellum were removed before the two hemispheres were separated from each other. Routinely, the right hemisphere or at times the whole brain was fixed via perfusion through the internal and basilar carotid arteries (whole brain), or the anterior, vertebral and carotid cerebral arteries (one hemisphere). A perfusion solution (PBS with 1% sodium nitrite pH 7.4) was flushed through the brain or hemisphere for 15 minutes and was then followed by a fixative containing 15% formalin in 0.1 M phosphate buffer for approximately 40 minutes. The brains or single hemispheres were postfixed for a 24-hour period in the same fixative, and then cut into blocks according to experimental interest. The dissected blocks were postfixed in 15% formalin solution for 24 hours, before being transferred to a 20% sucrose solution for 1 week, then a 30% sucrose solution for a further 2 to 3 weeks. The tissue blocks were then snap-frozen with powdered dry ice, wrapped in aluminium foil, coded, and stored at −80°C until required (H. J. Waldvogel, Curtis, Baer, Rees, & Faull, 2006). To acquire sections for immunohistochemical analysis, the relevant blocks were fixed onto a freezing microtome using OCT compound, and sections were cut at 70 μm and stored at 4°C in PBS-azide until being processed (H. J. Waldvogel et al., 2008; H. J. Waldvogel et al., 2006). For this study, sections from the middle region of the striatum located anterior to the anterior commissure were selected. For Western blotting procedures, the unfixed left hemisphere was dissected in matching anatomical blocks after the separation of the two hemispheres and was frozen immediately.

2.2. Antibody characterization

Primary antibodies used in this study are described in Table 2.

Table 2:

Antibodies used for immunoperoxidase and immunofluorescence labelling.

Antigen	Immunogen	Host Species	Dilution	Company/lot#
Calbindin D-28k (Calb)	Recombinant rat calbindin D-28k	Rabbit	1:5,000 (DAB) 1:2,000 (fluorescent)	Swant; CB-38a; RRID: AB_10000340
DARPP-32	PPP1R1B full length recombinant protein	Mouse	1:2,000 (DAB) 1:1,000 (fluorescent) 1:1000 (Western blot)	Creative Diagnostics; CABT-22918MH; RRID: AB_2472670
DARPP-32	Internal region of human origin DARPP-32	Goat	1:500 (DAB) 1:100 (fluorescent)	Santa Cruz; sc-31519; RRID: AB_639001
DARPP-32	Synthetic peptide corresponding to residues surrounding Glu160 of human DARPP-32	Rabbit	1:1000 (DAB)	Cell Signaling; 2306; RRID: AB_823479
Enkephalin (Enk)	Synthetic Leucine Enkephalin	Mouse	1:20,000 (DAB) 1:10,000 (fluorescent)	Seralab; MAS083c; RRID: N/A
Calretinin	Recombinant whole human calretinin	Rabbit	1:1,000 (fluorescent)	Swant; CR7699/3H; RRID: AB_10000320
Neuropeptide Y (NPY)	Synthetic whole porcine Neuropeptide Y	Rabbit	1:10,000 (fluorescent)	Sigma-Aldrich; N9528; RRID: AB_260814
Parvalbumin (Parv)	Purified carp muscle	Mouse	1:2,000 (fluorescent)	Swant; PV235; RRID: AB_10000343
Tyrosine Hydroxylase (TH)	Purified tyrosine hydroxylase from a rat pheochromocytoma	Mouse	1:2,000 (fluorescent)	Millipore; MAB5280; RRID: AB_90755
NeuN	GST-tagged recombinant protein corresponding to the N-terminus of mouse NeuN	Chicken	1:1000 (fluorescent)	Merck-Millipore; ABN91; RRID: AB_11205760

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Calbindin D-28k (Swant, Bellinzona, Switzerland, Cat. No. CB-38a, RRID: AB_10000340). Polyclonal rabbit antibodies to calbindin (AB1778) were raised against recombinant rodent calbindin D-28k. Western blots of rodent brain lysates revealed a single 28 kDa band (Chalazonitis et al., 2008). The immunoreactivity of calbindin-positive neurons was similar to that previously reported results (Sang & Young, 1998).

DARPP-32 (Creative Diagnostics, New York, U.S.A., Cat. No. CABT-22918MH, RRID: AB_2472670). This mouse monoclonal antibody was raised against a full length recombinant PPP1R1B (manufacturer’s product sheet).

DARPP-32 (Santa Cruz Biotechnology, Santa Cruz, California, U.S.A., Cat. No. sc-31519, RRID: AB_639001). This polyclonal goat antibody was targeted against a peptide mapping within an internal region of the human DARPP-32 (manufacturer’s product sheet). According to the datasheet, Western blotting techniques show the antibody to identify bands that correspond to the molecular weights and charge of DARPP-32.

DARPP-32 (Cell Signaling, Danvers, Massachusetts, Cat. No. 2306, RRID: AB_823479). This monoclonal rabbit antibody was produced by the immunization of animals with a synthetic peptide that corresponded to residues that surround Glu160 of the human DARPP-32. Western blots showed the antibody to identify a band (32 kDa) in wild-type mouse striatum. No band was identified in the knockout mouse striatum extract (manufacturer’s product sheet). The immunoreactivity identified by this antibody in rodent tissue is consistent with previous reports (Jeon et al., 2016; Yoshioka et al., 2011).

Enkephalin (Seralab, Sussex, United Kingdom, Cat. No. MAS083c, RRID: N/A). This monoclonal leu-enkephalin antibody was raised in mouse and targeted against synthetic leucine enkephalin. This particular antibody has been used reliably to identify enkephalin in post-mortem human tissue (Allen, Waldvogel, Glass, & Faull, 2009; Ekblad, Arnbjörnsson, Ekman, Håkanson, & Sundler, 1989; Tippett et al., 2007).

Calretinin (Swant, Bellinzona, Switzerland, Cat. No. CR7699/3H, RRID: AB_10000320). This polyclonal antibody was raised in rabbits against full-length recombinant human calretinin. In previous Western blots in tissue across a variety of species (rabbit, rat, chicken; manufacturer’s data sheet), the antibody was shown to be selective for a single band of 29–30 kDa (Schwaller, Brückner, Celio, & Härtig, 1999). Immunohistochemical results with the same antibody in knockout mice indicated null calretinin immunoreactivity (Bearzatto et al., 2006; Gall et al., 2005). Calretinin immunostaining distribution in human post-mortem brain tissue was consistent with previously reported findings (Cicchetti, Prensa, Wu, & Parent, 2000; H. Waldvogel et al., 1999).

Neuropeptide Y (NPY) (Sigma-Aldrich, St. Louis, Missouri, Cat. No. N9528, RRID: AB_260814). The polyclonal antibody was raised in rabbit against synthetic porcine NPY. The antiserum has been tested for cross reactivity with a variety of peptides including PYY, vasoactive intestinal peptide, and insulin, and has been proven to be specific for NPY (manufacturer’s certificate of analysis). The antibody has shown the expected staining patterns in spinal cord of rat and mouse (Corness, Shi, Xu, Brulet, & Hökfelt, 1996; Shehab, Spike, & Todd, 2003). There was no staining when the rat brain tissue was preincubated with the immunizing peptide (Sigma-Aldrich, Cat. No. N-5017, manufacturer’s technical information) (Real, Heredia, del Carmen Labrador, Dávila, & Guirado, 2009). Staining was in register with previous work conducted in post-mortem human tissue, and other species (Kowall et al., 1987; Y. Smith & Parent, 1986).

Parvalbumin (Swant, Bellinzona, Switzerland, Cat. No. PV235, RRID: AB_10000343). This mouse monoclonal antibody was raised against recombinant carp muscle parvalbumin. 2D immunoblots revealed specific staining for this antibody in the 45Ca-binding band of parvalbumin (manufacturer’s product sheet) (M. R. Celio, Baier, Schärer, De Viragh, & Gerday, 1988). The distribution of parvalbumin positive neurons that we observed was consistent with previous reports from rhesus and macaque monkeys (Bunce, Zikopoulos, Feinberg, & Barbas, 2013; Saleem & Logothetis, 2012).

Tyrosine hydroxylase (TH) (Merck-Millipore, Billerica, Massachusetts, Cat. No. MAB5280, RRID: AB_90755). This mouse monoclonal antibody identifies a band at ~64 kDa in Western blots of rat tissue. Furthermore, the antibody positively stains rodent sympathetic nerve terminals (manufacturer’s product sheet) (Cui, Su, Cao, Ma, & Qiu, 2021; Middeldorp, van den Berge, Aronica, Speijer, & Hol, 2009; Penkowa, Nielsen, Hidalgo, Bernth, & Moos, 1999; A. M. Smith et al., 2013).

Neuronal nuclear protein (NeuN) (Merck-Millipore, Billerica, Massachusetts, Cat. No. ABN91, RRID: AB_11205760). The polyclonal chicken antibody was raised against a GST-tagged recombinant protein corresponding to the first 97 amino acids from the N-terminal region of murine NeuN. The antibody recognizes the N-terminus of NeuN (manufacturer’s product sheet). This polyclonal chicken antibody identifies a band at ~48 kDa in Western blots of mouse brain E16 tissue lysate, according to the manufacturer’s data sheet. The staining was in register with previous work conducted in post-mortem human tissue and rat tissue (Mazur et al., 2019; Stevenson et al., 2020).

2.3. Western blotting

Fresh frozen human brain tissue was obtained from the Neurological Foundation Human Brain Bank. Samples of ~0.2 grams of total tissue weight for each case were dissected from blocks of fresh frozen striatal tissue from five cases (H241, H146, H194, H200, and H239.) Samples were individually placed in Eppendorf tubes with 1 mL of ice-cold PBS and centrifuged, and the solution was then aspirated to remove extraneous blood from the sample. One mL of homogenization buffer and silver beads were added to each tube, the tubes were then spun in the bullet blender and placed on ice for 1 hour, then spun at 14000 rpm for 10 minutes at 4°C. The supernatant was extracted from each tube and aliquoted for protein assays according to calculations to determine the volume of protein needed for gel electrophoresis and Western blotting with fluorescence detection. Protein solutions were made from homogenised protein samples, sample reducing agent, LDS sample, and MilliQ water. Samples were centrifuged and heated before running and transfer on an Invitrogen mini blot kit using a Bis-Tris gel system. Membranes were blocked with Odyssey blocking buffer and then incubated with primary and secondary antibodies. Membranes were then imaged using a LI-COR Odyssey imaging system.

2.4. Immunoprecipitation procedures

Magnetic beads (Bio-Rad SureBeads^™ Protein G Magnetic beads) were prewashed twice with 1 mL PBST buffer. Protein homogenates (combined from cases H200 and H239) were transferred to clear Eppendorf tubes (~1 mg protein/tube). One Eppendorf tube was allocated to test the mouse anti DARPP-32 antibody (Creative Diagnostics mouse anti DARPP-32 CABT-22918MH), while another was used as a control with a mouse IgG antibody (Vector Laboratories Mouse IgG Control Antibody I-2000). The prewashed magnetic beads were added to each Eppendorf tube to pre-clear any non-specific binding, the tubes were incubated at 4°C on a rotating wheel for 30 minutes, and the magnetic beads were separated from the supernatants with a magnetic rack, the supernatants were placed in clean Eppendorf tubes, and 10 μL of protease and phosphatase inhibitor (Thermo Scientific Halt^™ Protease and Phosphatase Inhibitor Cocktail) was added to each tube. Primary antibodies were added at 4 μg/1 mg of protein in each sample (8 μL of the mouse anti DARPP-32 antibody per trial tube, and 2 μL of the mouse control IgG antibody for the control tube). Tubes were incubated for 2 hours at 4°C on a rotating wheel, new magnetic beads were washed 2x using PBST before being added to the trial and control Eppendorf tubes, and all tubes were incubated overnight at 4°C on a rotating wheel. The magnetic beads were then separated from the supernatants and the supernatants were removed, the magnetic beads were washed 3x in PBST and all elutants were removed before the beads were transferred for mass spectrometry.

2.5. Immunohistochemical procedures

2.5.1. Immunoperoxidase labelling (select antibodies)

Selected sections of brain tissue were transferred from PBS-azide storage solution to tissue culture well plates, washed once in PBS and 0.2% Triton-X (PBS-triton), then incubated in fresh PBS-triton overnight at 4°C on a rocker and washed in PBS-triton for three 10-minute periods between each of the following steps. On the following day, the sections underwent a blocking step consisting of 20-minute incubation in 50% methanol with 1% H₂O₂ to block endogenous peroxidases and to reduce non-specific binding. Sections were then incubated with the relevant primary antibody diluted to optimal concentrations in 1% normal goat or donkey serum for a 48–72 hour period on a rocker at 4°C. Control sections were incubated in normal goat or donkey serum only at this stage. The following mouse, rabbit, and goat antibodies were used: rabbit anti calbindin D-28k (1:2000, Swant CB38a), mouse anti DARPP-32 (1:2000, Creative Diagnostics CABT-22918MH), goat anti DARPP-32 (1:500, Santa Cruz sc-31519), mouse anti enkephalin (1:20,000 Seralab MAS083c). Sections were washed in PBS-triton to remove unbound primary antibodies, and then incubated overnight in species-specific biotinylated secondary antibodies diluted in 1% normal goat or donkey serum at room temperature: goat anti-mouse (1:1000, B-7264 Sigma), goat anti-rabbit (1:2000, Sigma B-7389), donkey anti-goat (1:5000, Jackson 705-065-003,). Sections were then washed to remove unbound secondary antibodies, then incubated for 4 hours in ExtrAvidin diluted in the appropriate immunobuffer, washed again, and a [3,3’-diaminobenzidine-tetrahydrochloride] (DAB) in 0.1 M phosphate buffered solution was added together with 0.01% H₂O₂ for up to 20 min to achieve a brown reaction product. Sections were then washed in PBS-triton for three 10-minute washes, mounted onto gelatine chrom-alum coated slides, and left to dry for a week. The slides were then dehydrated through a graded alcohol series, and finally placed in Xylene for 1 hour. Mounting medium (DPX) was then applied, and coverslipped sections were left to dry completely before analysis (H. J. Waldvogel et al., 2006).

2.5.2. Immunoperoxidase labelling (Rabbit anti DARPP-32 only)

Fixed frozen sections were selected from storage and washed in PBS-T (0.01 M PBS + 0.2% Triton solution) for 5 minutes at room temperature. Sections were then added to the sodium citrate solution (10 mM sodium citrate solution (pH 8.5) brought to 75–80°C) and were incubated for 30 minutes. The solution was then left at room temperature for 10 minutes, the sections were washed again in PBS-T for 5 minutes on a plate rocker at room temperature, a peroxidase blocking solution was then applied to the sections for 20 minutes and the sections were again washed 3 times (10 minutes per wash) in PBS-T. Sections were blocked in 10% normal goat serum in PBS-T for 20–30 minutes, then incubated in rabbit anti DARPP-32 antibody (Cell Signaling; 2306) diluted in normal goat serum immunobuffer to a final antibody concentration of 1:1000 overnight at 4°C. The next day, sections were washed 3 times (10 minutes per wash) in PBS-T, then incubated in a goat anti-rabbit secondary antibody (conjugated to HRP) in normal goat serum buffer for 2–3 hours on a rocker at room temperature. A DAB solution was added to the sections for 5–20 minutes, sections were washed 3 × 5 min in PBS and mounted onto labelled slides in gelatine solution. These were left to dry, then taken through an alcohol dehydration series, and coverslipped.

To determine the degree of non-specific staining from secondary antibodies, primary-omission control sections (sections not incubated with primary antibodies but otherwise treated as in the standard protocols) were run in parallel for the mouse anti and goat anti DARPP-32 antibodies on human and rat brain tissue.

2.5.3. Immunofluorescence labelling

Sections were transferred from PBS-azide to tissue culture well plates containing PBS-triton, washed once, and incubated in PBS-triton overnight at 4°C. The next day, the sections were incubated in primary antibodies diluted at optimal concentrations in the appropriate species specific serum, for 48–72 hours at 4°C. If two primary antibodies were added to a particular section simultaneously for double labelling, it was ensured that the two antibodies were raised in different species. Control sections for double labelling included comparing sections with single staining for each primary antibody with sections stained with both secondary antibodies. Another control section was treated without primary antibodies but with both secondary antibodies to test for non-specific labelling or autofluorescence.

Following primary incubations, the sections were given three 10-minute washes in 1x PBS to remove unbound primary antibodies, fluorescently labelled secondary antibodies specific to the relevant primary antibodies diluted in 1% normal serum were then applied, and sections were incubated in the dark on a rocker at room temperature. The fluorescent secondary antibodies included anti-goat (1:500, Invitrogen) and anti-donkey (1:500, Dako) antibodies directly linked to Alexa fluorophores 488 and 594. The next day, the sections were again washed three times for 10 minutes each in 1x PBS to remove unbound secondary antibodies. Immediately afterward, the sections were counterstained for 20 minutes with Hoechst solution to label for cell nuclei. The sections were then washed for two 2-minute washes in PBS, and then were mounted immediately onto slides using 1x PBS. The slides were coverslipped with Prolong Gold as a mounting medium and were stored at 4°C in the dark overnight to dry. Nail varnish was then used to seal the edge of the coverslip to prevent the leaking of the mounting media, and again left to dry at 4°C overnight in the dark to dry. After this processing, the slides were imaged and analysed.

2.6. Imaging

2.6.1. Immunoperoxidase stained sections (selected antibodies)

The DAB-stained sections were viewed with a Leica brightfield microscope, and macroscopic and stacked digital images were captured with a Nikon D100 digital camera and saved in a Tiff format. The sections were also viewed with a Nikon Eclipse Ni upright light microscope. This microscope was coupled to a Nikon DS-Ri2 camera, which was used to capture photomicrographs.

2.6.2. Immunoperoxidase-stained sections (Rabbit anti DARPP-32 only)

The sections were examined and images of selected sections were captured with an Olympus DP70 Digital Camera mounted on an Olympus Bx61 upright light microscope. The digital images were likewise saved in a Tiff format.

2.6.3. Immunofluorescent-stained sections

The fluorescent sections used for double labelling were photographed using an Olympus FV1000 confocal laser scanning microscope and these images were saved in a Tiff format.

2.7. Cell counts

2.7.1. Manual cell counts on immunoperoxidase-stained human striatal sections for calculations of density

Striatal slices from control brains (cases H140, H160, H170, H186 and H242) were stained with DARPP-32 using DAB immunoperoxidase techniques. Images of the caudate area were acquired using the TissueFAXs slide scanner with a 20x objective. The size of each field of view (FOV) was calculated to be 2048 × 2048 pixel (480.75 μm × 480.75 μm). Then, 7 striosome FOVs and 7 matrix FOVs were selected from each sample. DARPP-32-positive cells were then counted manually, and cell density was calculated by dividing the number of cells by striosomes or matrix area (excluded blood vessels).

2.7.2. Automatic cell counts on immunofluorescent stained post-mortem human and rat striatal sections

For this section of analysis, we examined the proportion of the neuronal population in the human and rat caudate nucleus that expressed either only DARPP-32, only calbindin, or both DARPP-32 and calbindin. Multi-immunofluorescent labelling was performed using mouse anti DARPP-32, and rabbit anti calbindin. Chicken anti NeuN was used as a pan neuronal marker. Four post-mortem human brain cases were chosen (H160, H186, H241, and H242) and two striatal sections were chosen from each case. Furthermore, two striatal sections were selected from four post-mortem rat brain cases each.

After multi-immunofluorescent labelling was performed, a Zeiss inverted confocal microscope (LSM-710 inverted confocal laser scanning microscope) was used to capture multi-channel images from five striosomal regions and five matrix regions from each human striatal section (it is to be noted that the striosomal and matrix regions were only selected from the dorsal caudate nucleus) with a 20x objective. The size of each FOV was calculated to be 1024 × 1024 pixel (425.10 μm × 425.10 μm). Multi-channel images were also captured from the rat striatum (from the five striosomal regions and five matrix regions). Within the set of images captured from both human and rat striatal tissue, striosomes were identified as regions exhibiting weak calbindin stain, and the matrix as calbindin-rich zones. All images were exported as 8-bit TIFFs for analysis using FIJI software (version 1.53).

For the human striatal sections, the images containing NeuN staining were processed using a median filter (Ω = 3) and Gaussian filter (Ω = 30) applied to duplicates of the original image. The Gaussian processed image was subtracted from the median processed image to reduce background noise and to identify the NeuN-positive cells. The image result was auto-thresholded using the ‘Default’ method and converted to a mask. The mask was slightly dilated (iterations = 4, count = 1), before the image was made binary and a watershed threshold was run again. Particles were analysed and regions of interest (ROI) were logged using the ROI manager and saved.

Next, the images containing Hoechst (cell nuclear marker), calbindin and DARPP-32 were processed sequentially by duplicating the images to make the images suitable for processing using a median filter (Ω = 2 for all) and to make one more identical image suitable for Gaussian filtering (Ω = 45 for Hoechst, Ω = 35 for calbindin, Ω = 30 for DARPP-32). The Gaussian filtered image was subtracted from the median filtered image to create a mask to reduce background noise and to identify the cell nuclei more clearly. The image result was then auto-thresholded using the ‘Li’ method (Hoechst), the ‘Otsu’ method (calbindin) or the ‘Default’ method (DARPP-32) and converted to a mask. The mask was made binary before watershed thresholding was performed. As with NeuN, the analyse particles function was run to obtain a final binary image.

The NeuN ROI mask was overlaid onto each binary image corresponding to each marker. NeuN ROIs were measured using the ‘measure regions’ function in FIJI software in the ROI manager. The results were then saved into Microsoft Excel spreadsheets. Neuronal cells as marked by NeuN were counted using automated methods in Excel and assigned to one of three categories (DARPP-32 only positive, calbindin only positive, or cells with DARPP-32 + calbindin colocalised). Our reference cells were the NeuN cells, and provided that a NeuN cell exhibited Hoechst, DARPP-32 and/or calbindin, it was incorporated into our analyses.

In order to calculate cell population proportions for the dorsal human matrix compartments, cells were summed across all the matrix positions for each human case, and then summed again across all four human cases. Cell percentages were calculated as a percentage of all NeuN-positive cells, and also NeuN-positive SPN-like cells that showed DARPP-32 and/or calbindin immunoreactivity. The same analyses and methods were followed for cell proportions in the dorsal human striosomes.

For the sections through the striatum of the rats, the images containing NeuN staining were thresholded using a median filter (Ω = 2) and Gaussian filter (Ω = 15) applied to original image duplicates. The image subtract function was followed as previously mentioned for the purposes of obtaining an image result, and to identify the NeuN-positive cells. A mask was created by the image result being auto-thresholded using the ‘Otsu’ method. Regions of interest were logged using the ROI manager and saved.

Next, the images containing Hoechst and calbindin were duplicated to make one image suitable for processing using a median filter (Ω = 2 for all) and to make another identical image suitable for Gaussian filtering (Ω = 18 for Hoechst, Ω = 25 for calbindin). The Gaussian filtered image was subtracted from the median filtered image to create a mask to reduce background noise, and to identify the cell nuclei as appropriate. Auto-thresholding was conducted using the ‘Huang’ method (Hoechst), or the ‘Yen’ method (calbindin) on the image result, and converted to a mask. This mask was converted to a binary image before watershed thresholding was performed. As with NeuN, the analyse particles function was run to obtain a final binary image.

The NeuN ROIs were then overlaid on the Hoechst and calbindin binary images. Due to the high neuropil staining of DARPP-32, we applied the NeuN ROI outlines to the raw images of DARPP-32 and measured the intensity of DARPP-32 staining. Through pilot runs conducted on each case, we determined minimum threshold intensities for DARPP-32 cells and applied these measures for automated counting.

The ‘measure regions’ function was utilised in FIJI software. The results were then saved into Microsoft Excel spreadsheets, and from here, using NeuN ROI as a base, any ROI that showed colocalization with Hoechst was counted as a positive NeuN cell. After this, cells were counted using automated methods in Excel and assigned to one of three categories (DARPP-32 only positive, calbindin only positive, or cells with DARPP-32 + calbindin colocalised). As previously stated, provided that a NeuN cell exhibited Hoechst, DARPP-32 and/or calbindin, it was incorporated into our analyses.

For the calculations of cell population proportions for the rat matrix compartments, cells were summed across all the matrix positions for each rat case, and then summed again across the four rat cases. Cell percentages were calculated as a percentage of all NeuN-positive cells, and additionally NeuN-positive SPN-like cells that showed DARPP-32 and/or calbindin immunoreactivity. Furthermore, the same analyses methods were conducted for cell proportions in the rat striosome compartment.

3. RESULTS.

3.1. Validation of the DARPP-32 antibodies with Western blotting techniques

The monoclonal mouse anti DARPP-32 antibody (Creative Diagnostics; CABT-22918MH) and the polyclonal goat anti DARPP-32 antibody (Santa Cruz; sc-31519) exhibited two specific bands in Western blots from human striatal tissue homogenates (Figure 1a and b). The two bands identified by both antibodies represent a DARPP-32 doublet, with the higher band corresponding to 32 kDa (fitting the weight of DARPP-32 protein), and the lower band corresponding to the weight of [phosphor-Ser-137] DARPP-32, interpreted as corresponding to a human isomer of DARPP-32 phosphorylated at Ser-137 and Thr-34. This interpretation is consistent with previous representation of DARPP-32 in Western blots (Frédéric Desdouits, Cohen, Nairn, Greengard, & Girault, 1995; Frederic Desdouits, Siciliano, Greengard, & Girault, 1995). The rabbit monoclonal DARPP-32 antibody (Cell Signaling; 2306) has been validated in rat and mouse homogenates using Western blotting, and, in addition, by use of extracts from a DARPP-32 knockout mouse yielding no labelling by the rabbit DARPP-32 antibody in Western blots (manufacturer’s datasheet) (data not shown).

3.2. Mass spectrometry analysis of Western blot and immunoprecipitation samples

The lower and upper bands identified by Western blot analysis with the anti-mouse DARPP-32 antibody (Figure 1a) were separately selected and analysed with mass spectrometry performed by the Mass Spectrometry Centre, The University of Auckland, Auckland, New Zealand. DARPP-32 (listed as protein phosphatase 1 regulatory subunit 1B, or PPP1R1B) was present in both bands, but at low levels. As the regions of the Western blot membrane excised for mass spectrometry were large to prevent missing the DARPP-32 bands, it was not unusual that other proteins would have been present in the membrane areas processed for mass spectrometry. Following this initial test, an enrichment procedure was performed by immunoprecipitation, and mass spectrometry was performed again on a single sample containing magnetic beads processed with homogenates from fresh frozen human striatal tissue and the mouse anti DARPP-32 antibody (Creative Diagnostics anti-mouse DARPP-32 CABT-22918MH). A control sample containing magnetic beads processed with homogenates from fresh frozen human striatal tissue and a generic mouse IgG antibody (Vector Laboratories Mouse IgG Control Antibody I-2000) was processed for comparison. DARPP-32 was present in the test sample but was not present in the control sample. The table in Figure 1c indicates the total protein identification scores for the top 5 proteins obtained via mass spectrometric analysis of the DARPP-32 positive and control mouse IgG immunoprecipitated samples. As DARPP-32 (Protein Phosphatase 1 regulatory subunit 1B; PPP1R1B) was in the top 3 proteins, we have confidence that the mouse anti DARPP-32 antibody correctly identifies the DARPP-32 protein in post-mortem human tissue.

3.3. Consistency of immunostaining patterns and comparison of mouse and human striatal DARPP-32 immunostaining patterns

The primary antibody omission human and rat striatal sections, for which the monoclonal mouse anti DARPP-32 antibody (Creative Diagnostics; CABT-22918MH) and polyclonal goat anti DARPP-32 antibody (Santa Cruz; sc-31519) were omitted (Figure 1e, g, i, k), lacked antibody-specific immunostaining, confirming that the standard immunohistochemical staining with primary antibodies present was not due to non-specific secondary antibody immunoreactivity (Figure 1d–k). Multiple immunohistochemical experiments were conducted with all three antibodies (mouse anti DARPP-32, goat anti DARPP-32, and rabbit anti DARPP-32) on the human tissue sections and on rat tissue sections with the mouse and goat anti-DARPP-32 antibodies, and all tested antibodies evoked similar species-specific staining patterns at both the regional and cellular levels. The Creative Diagnostics mouse monoclonal DARPP-32 antibody and the Santa Cruz goat polyclonal DARPP-32 antibody were the main antibodies used for this publication.

Sections from the dorsal striatum of the human (Figure 2a–f) exhibited a marked patchy staining pattern reminiscent of striosome/matrix patterning following immunostaining with each of the three (mouse, goat and rabbit) anti DARPP-32 antibodies. By contrast, patterns of immunostaining in sections through the caudoputamen of the rat brains stained with these same anti DARPP-32 antibodies indicated relatively homogeneous staining for DARPP-32, with some preference of staining for the matrix. Staining of rat sections with mouse anti and goat anti DARPP-32, and calbindin revealed differences between DARPP-32 and calbindin representation in the rat striatum, with DARPP-32 showing homogeneous staining throughout the striosomes and the matrix, whereas calbindin was high in the matrix but low in striosomal zones and lateral crescent of the caudoputamen, confirming earlier work (M. Celio, 1990) (Figure 2g–j).

3.4. DARPP-32 immunolabelling of neuropil in the dorsal human striatum was predominantly located in striosomes

Serial-section immunostaining of the human striatum revealed intense DARPP-32 immunoreactivity in striosomes compared to matrix particularly in the dorsal human striatum (Figures 2a, b, 3a, b). The DARPP-32-positive regions corresponded to striosomes as identified in adjacent calbindin-stained sections by their low calbindin expression of neuropil and cell bodies (Figure 3c, d). The DARPP-32 striosomes were particularly striking in the dorsal caudate nucleus and adjoining dorsomedial putamen. Corresponding striosomal regions were also identified in serially adjoining enkephalin-immunostained sections, which exhibited characteristic enhanced enkephalin immunoreactivity in the neuropil of the striosomes relative to that in the adjoining matrix (Figure 3e, f).

Figure 3. — Macroscopic photomicrographs of serial sections of the normal human striatum stained for DARPP-32 (a and b), calbindin (matrix marker; c and d), and enkephalin (striosomal marker; e and f). The macroscopic photomicrographs indicate the presence of increased DARPP-32 staining of the striosomes (arrows in a). The striosomal staining is particularly obvious in the caudate nucleus (a) where a striosome is indicated by the black box showing increased DARPP-32 stain; the same striosome is indicated by black boxes with decreased calbindin stain (c) and increased enkephalin stain (e), respectively confirming that the patches of increased DARPP-32 staining are located in striosome. This particular striosome is shown at a greater magnification in b, d and f as serial sections stained with DARPP-32 (a), calbindin (c) and enkephalin (e). A single striosome was noted and higher magnification images were captured of this striosome from all three striatal sections. Dense DARPP-32 immunoreactivity is present in the neuropil and cell bodies of the striosome (b). The same striosome shows a characteristic lack of calbindin immunoreactivity (d) and moderate enkephalin immunoreactivity (f). All images are from human brain case H242. AC, Anterior commissure; CN, caudate nucleus; GP, globus pallidus; IC, internal capsule; Put, putamen; VS, ventral striatum. Scale bars: 0.5 cm (a,c,e), 500 μm (b,d,f)

3.5. Striosome and matrix architecture in the normal human dorsal striatum: view at pre-anterior commissural levels with immunofluorescent staining for DARPP-32, calbindin and enkephalin, with comparisons to rodent patterns.

Striosomes within the caudate nucleus at low magnification (Figure 4a–d) exhibited intense DARPP-32 immunoreactivity of the cell bodies and neuropil (Figure 4a). Confocal images of the same region using different markers showed a calbindin-poor zone (Figure 4b) and an enkephalin-rich striosomal area (Figure 4c) of the same annotated region. When overlaid with the calbindin and enkephalin immunostaining (Figure 4d), the DARPP-32-rich striosome showed an overlap with the calbindin-poor zone and the enkephalin-rich striosomal area. The high magnifications of the striosome border (Figure 4e–i) provide a closer view of the immunostaining profiles, illustrating increased DARPP-32 immunoreactivity within the cells and neuropil of the striosome as contrasted with the low immunostaining of the adjoining matrix region (Figure 4e). Conversely, the striosome showed low calbindin immunoreactivity within the cells and neuropil (Figure 4f).

In the human putamen, DARPP-32 immunolabelling also identified the striosome compartment in complementary fashion to calbindin immunolabeling (Figure 5b), but in conjunction with enkephalin (Figure 5c). An enkephalin-poor zone (indicating the striosome core) was surrounded by a rim of high enkephalin immunostaining that indicated the borders of the striosome (Figure 5c). The ‘core’ of the striosomes has been mentioned in previous literature (Brimblecombe & Cragg, 2017; Holt et al., 1997; Liu & Graybiel, 1992; Matsushima & Graybiel, 2020; Prensa, Giménez-Amaya, & Parent, 1999), and it has been suggested that the core of the striosome receives a concentrated amount of dopaminergic innervation from the substantia nigra (Prensa et al., 1999). Higher magnifications of the striosome border in the post-mortem dorsal human putamen indicate that, as in the post-mortem human caudate nucleus, increased DARPP-32 immunoreactivity is present in cells and neuropil of the striosome (Figure 5e), and conversely, increased calbindin immunoreactivity is seen in the cells and neuropil of the matrix (Figure 5f). The merged image (Figure 5i) shows a clear difference of DARPP-32 immunostaining and calbindin immunostaining between the striosome and matrix compartments. These results confirm that a given section through the striosomal system in the normal dorsal human striatum could have differing apparent boundaries depending on the marker of interest.

Figure 5. — Striosome borders in the normal human dorsal putamen as viewed with immunofluorescent staining of DARPP-32, calbindin and enkephalin. (a-d) Low power photomicrographs of immunofluorescent stained human caudate nucleus with DARPP-32 (a), calbindin (b) and enkephalin (c), concentrated on a single striosome (region enclosed by cyan dashed line). DARPP-32 primarily shows high immunoreactivity in the striosome (a). However, by comparison, calbindin immunoreactivity of the same striosome shows low immunoreactivity in the core of the striosome only (b). Enkephalin shows a ring of enkephalin immunoreactivity in the peripheral zone of the striosome (c). In the merged channels (d), it is seen that the three different markers do not consistently define the same striosomal core boundaries, although they all identify the core of the striosome to some extent. (e-i) High magnification photomicrographs of a portion of the striosome and matrix including the striosome border, as indicated by the white dashed box in d. The high magnification images indicate DARPP-32 (e), calbindin (f), enkephalin (g), Hoechst (h), and the final merged image (i). As with the human dorsal caudate nucleus, a high amount of DARPP-32 is observed in the cells and neuropil of the striosome (S), with limited DARPP-32 immunoreactivity in the matrix (M). Calbindin shows some increased immunoreactivity in the neuropil of the matrix (f). The merged image in particular shows a very different distribution of DARPP-32 and calbindin across the striosome/matrix border (i). Scale bars: 200 μm (a-d), 50 μm (e-j)

The fluorescence staining in the rat striatum shows a different picture regarding striosomes. While DARPP-32 in the rat striatum shows a homogenous distribution amongst cells and neuropil (Figure 6a), there is a notable low level of calbindin staining in the striosome (Figure 6b). The evidence that this region is indeed a striosome is corroborated by an increased amount of enkephalin within the striosome region (Figure 6c). The merged image of DARPP-32 and calbindin (Figure 6d) shows a distinct green shadow (indicating DARPP-32 immunoreactivity within the striosome), amongst a background of magenta neuropil and cells (indicating calbindin immunoreactivity within the matrix). The conclusion from the rat work is that while DARPP-32 is indeed present within the striosomes in the rat striatum, the immunoreactivity of DARPP-32 within the cells and neuropil of the striosomes is not vastly different from DARPP-32 immunoreactivity within the cells and neuropil matrix, as opposed to results seen in the human striatum.

Figure 6. — Immunofluorescent staining of DARPP-32, calbindin and enkephalin in the rat striatum. There was no distinguishable difference in DARPP-32 immunoreactivity regarding the cells and neuropil within the striosome and matrix (a), as opposed to calbindin (b) and enkephalin (c). A single striosome is delineated by a blue dashed line. The distribution of DARPP-32 appears to be fairly homogenous across both the striosome and matrix compartments. The merged image shows DARPP-32 immunoreactivity within the striosome, but limited calbindin immunoreactivity within the striosome. Scale bars: 100 μm

3.6. In the human striatum, DARPP-32-positive striosomal borders are more distinct in the caudate nucleus than in most of the putamen except at rostral levels

To illustrate the different mediolateral and anteroposterior gradients in DARPP-32 immunostaining of the striatum, we analysed rostral-to-caudal series of striatal sections from three post-mortem human brain cases stained with DARPP-32, making qualitative assessments of the clarity and definition of striosome borders in the caudate nucleus and the putamen. In the most rostral parts of the striatum, striosomes were not as obvious as viewed at more caudal levels through the head of the caudate nucleus (Figure 7a, f, k). However, at levels at which both the caudate nucleus and the putamen were present in the coronal view of the striatum, the DARPP-32-positive striosomes in the caudate nucleus were definite and possessed distinct borders by comparison to DARPP-32-positive striosomes in most of the putamen (Figure 7b, g, l). Only near the internal capsule, and dorsally, were there crisply shown DARRP-32-positive striosomes detectable. This pattern was consistent across the selected cases and continued at least as far caudally as the level of the globus pallidus (Figure 7i, j, n), the most caudal levels studied. While case variability can play a factor in the immunoreactivity of DARPP-32 in various human brain cases, the ability of DARPP-32 to identify striosomes in the dorsal striatum, in both the cells and neuropil, and in contrast to the surrounding matrix, proved to be consistent across all the human brain cases examined in this study.

Figure 7. — Photomicrographs of basal ganglia sections from three different normal cases (H160, H215 and H242), arranged rostrally to caudally. Across all cases, DARPP-32-positive striosomes are more distinct in the caudate nucleus regions, in opposition to the neighboring putamen regions. The DARPP-32-positive striosomes in the putamen are less vivid and distinct. It is thus postulated that striosomes are more weakly present in putamen regions. It is noted that DARPP-32 also exhibits immunoreactivity in the globus pallidus regions (GPe and GPi). Images in a-e are from human brain case H160, images in f-j are from human brain case H215, and images in k-o are from human brain case H242. HCN, head of caudate nucleus; CN, caudate nucleus; Put, putamen; TCN, tail of caudate nucleus; GPe, globus pallidus externa; GPi, globus pallidus interna; AC, anterior commissure. Scale bars: 0.5 cm

3.7. Immunodetectable DARPP-32 was localized in cells resembling SPNs in the caudate nucleus of the human striatum

Within the striosomes, the density of DARPP-32-positive neuropil staining made it difficult to distinguish individual cell bodies and their processes (Figure 8a, c). We did succeed, however, in identifying within the striosomes densely defined DARPP-32-positive neurons visible by virtue of their darker staining (Figure 8c) as opposed to the surrounding matrix neurons (Figure 8b). These DARPP-32-positive neurons possessed cell bodies that ranged from 10–15 μm in diameter, with spiny dendrites. (Figure 8a, b).

Figure 8. — Higher magnification photomicrographs of the striosome from human brain case H242 outlined by the black box in Figure 3. (a) Increased concentration in the number of DARPP-32-positive cells in the striosomes (S), as well as increased staining in the striosomal neuropil. By contrast, in the matrix compartment (M), the distribution of DARPP-32-positive cells and staining of DARPP-32 in the neuropil was markedly less. (b) A higher magnification image of a DARPP-32-positive neuron in the matrix (indicated by the box with dashed black lines in a) showing a spiny dendritic morphology. (c) A higher magnification of DARPP-32-positive cells in the striosome compartment (indicated by the box with the solid black line in a). Comparison of b and c shows that the striosome has an increased DARPP-32 cell number and increased DARPP-32 neuropil immunoreactivity, compared with the matrix (arrowheads in b and c indicate spines). Scale bars: 50 μm (a), 10 μm (b and c)

Despite the overall weaker DARPP-32 immunostaining of the matrix compartment, there were many DARPP-32-positive cells. Some of these had primary dendrites that exhibited a smooth contour proximally and apparent spines on the more distal dendritic branches (secondary- and tertiary-order dendrites) (Figure 8b). At a higher magnification, these neurons were observed to have spine-rich dendrites. The morphologies of these spines presented a mixture of stubby and mushroom-like spines. The cell bodies displayed a variety of shapes (spheroid, ovoid and pyramidal) and polarization (unipolar, bipolar, and multipolar).

3.8. Higher density of DARPP-32-positive cell bodies in striosomes than the matrix of the dorsal human caudate nucleus

To estimate the density of clearly DARPP-32-immunoreactive neurons in striosomes and matrix of the human striatum, we took brightfield images of entire sections through the dorsal caudate nucleus (Figure 9a). Seven samples with identical FOVs of striosomes (Figure 9b) and matrix (Figure 9c) were selected. DARPP-32-positive cells were manually counted, and the distribution densities of the DARPP-32-positive cells were calculated by dividing the number of cells by the area of striosomes and matrix (Figure 9d). As shown in Figure 9d, the density of DARPP-32-positive cells was higher in striosomes than the density in the matrix (p < 0.0008, t-test).

Figure 9. — DARPP-32-positive cell count from caudate nucleus. (a-c) Tiled photomicrograph of DARPP-32 (a), and one FOV from striosome (b) and matrix (c). (d) Number of DARPP-32-positive cells divided by striosomes or matrix area. The density of DARPP-32-positive cells was higher in striosomes than matrix (*p < 0.0008, t-test). Error bars show SEM. Scale bars: 1 mm (a), 50 μm (b and c)

3.9. Within the dorsal striatum of the human, DARPP-32 was not detectable in the striatal interneurons identified

We obtained reliable dual-antigen immunostaining of DARPP-32 positive cell soma alongside parvalbumin-immunoreactive cells (Figure 10a), calretinin-immunoreactive cells (Figure 10b), and NPY-immunoreactive cells (Figure 10c), each type considered to be interneuronal based on their co-localized peptide. We did not detect double labelling for DARRP-32 in any of these cell types (Figure 10). Nor did the morphology of the DARPP-32-positive cell resemble that described for these interneuronal types (Cicchetti et al., 2000; Y. Smith & Parent, 1986). We also did not detect tyrosine hydroxylase (TH)-immunostaining in DARPP-32-positive fibres, but TH-positive terminals did appear close to some DARPP-32-positive neurons (Figure 10d). The images illustrated in Figure 10 were captured from regions of the matrix compartment, but the same held true for the striosomes examined.

3.10. Double-immunofluorescent labelling of DARPP-32 and calbindin suggest the presence of three SPN or SPN-like cell populations in the dorsal striatum of the human and rat

Qualitative examinations of double-immunofluorescent labelling indicated that in the human dorsal striatum DARPP-32 and calbindin do not show complete colocalization in all of the SPNs examined in both the striosome and matrix compartments. We could classify separate SPN populations based on their immunostaining characteristics: a DARPP-32-only population, a calbindin-only population, and the third population showing co-localization of DARPP-32 and calbindin. These three SPN populations were observed both in striosomes (Figure 11a–c) and in matrix (Figure 11d–f). These three cell populations shared the characteristic appearance of SPNs (Braak & Braak, 1982; Graveland, Williams, & Difiglia, 1985). The images in Figure 11a–f were captured from the dorsal part of the caudate nucleus; we observed similar SPN sub-types in the dorsal human.

Observations in the rat striatum indicate that a large number of putative SPNs were stained with DARPP-32 (Figure 11g) in both the striosome and matrix compartments, and most of the cells in the matrix were stained with calbindin (Figure 11h). A large proportion of the cells appear to show colocalization for both DARPP-32 and calbindin, as seen in the merged image (Figure 11i), but we also observed that a few scattered cells are DARPP-32-positive only. There were few calbindin-only SPN-like cells in the rat striatum.

Automated cell counts in the human and rat striatum made for three separate SPN-like populations (i) DARPP-32 positive only (ii) calbindin-positive only (iii) DARPP-32 and calbindin positive in reference to a total NeuN-positive cell population conducted across the striosome and matrix compartments demonstrated that these three cell populations were present in both the human and rat striatum, but with different proportions in each species.

In the dorsal human striatum (n = 4, 2 sections per case), the total populations of NeuN cells were similar in the striosomes and matrix (Table 3), and the cell proportions of NeuN cells exhibiting co-localization of DARPP-32, calbindin, and both DARPP-32 and calbindin were different in the striosome and matrix compartments (Table 3, Figure 12a). The NeuN/DARPP-32 and NeuN/DARPP-32/calbindin cell populations in the human striatum were higher in the striosomes than the dorsal human matrix, but conversely, the NeuN/calbindin cell population was higher in the matrix than in the striosomes (Figure 12a).

Table 3:

Mean and total cell counts of the three cell types based on calbindin and DARPP-32 reactivity within the striosomes and the matrix of the dorsal human caudate nucleus (n = 4) and the dorsal rat striatum (n=4).

Human Striosomes	Case Mean Cell Numbers
	H160	H186	H241	H242	Mean	Percentage
NeuN only	56.2	54.3	39.6	45.4	48.88	61.56
NeuN/DARPP-32	14	9.9	13.6	9.4	11.725	14.77
NeuN/Calb	17.5	14.8	5.1	14.6	13	16.37
NeuN/DARPP-32/Calb	5.8	4.6	5.9	6.9	5.8	7.30

	Case Total Cell Numbers
	H160	H186	H241	H242	Sum	Percentage of Sum Total
NeuN/DARPP-32	140	99	136	94	469	38.41
NeuN/Calb	175	148	51	146	520	42.59
NeuN/DARPP-32/Calb	58	46	59	69	232	19.00

Human Matrix	Case Mean Cell Numbers
	H160	H186	H241	H242	Mean	Percentage
NeuN only	44.9	50.7	39.4	31.9	41.725	58.81
NeuN/DARPP-32	4.8	9.8	9.6	4.5	7.175	10.11
NeuN/Calb	25.2	14.5	13.4	18.9	18	25.37
NeuN/DARPP-32/Calb	3.1	4.4	6.6	2.1	4.05	5.71

	Case Total Cell Numbers
	H160	H186	H241	H242	Sum	Percentage of Sum Total
NeuN/DARPP-32	48	98	96	45	287	24.55
NeuN/Calb	252	145	134	189	720	61.59
NeuN/DARPP-32/Calb	31	44	66	21	162	13.86

Rat Striosomes	Case Mean Cell Numbers
	DY64	DY66	DY67	DY68	Mean	Percentage
NeuN only	9.3	5.5	6.8	10	7.9	12.83
NeuN/DARPP-32	62.4	40.1	41	49.1	48.15	78.20
NeuN/Calb	0.9	0.5	0.3	0.4	0.525	0.85
NeuN/DARPP-32/Calb	5.7	3.2	8.5	2.6	5	8.12

	Case Total Cell Numbers
	DY64	DY66	DY67	DY68	Sum	Percentage of Sum Total
NeuN/DARPP-32	624	401	410	491	1926	89.71
NeuN/Calb	9	5	3	4	21	0.98
NeuN/DARPP-32/Calb	57	32	85	26	200	9.32

Rat Matrix	Case Mean Cell Numbers
	DY64	DY66	DY67	DY68	Mean	Percentage
NeuN only	11.7	9.78	0	18.78	10.06	12.64
NeuN/DARPP-32	19.9	25.33	24.44	12.22	20.475	25.72
NeuN/Calb	7.1	0.33	0	8.56	3.99	5.02
NeuN/DARPP-32/Calb	49	41.11	50	40.22	45.08	56.62

	Case Total Cell Numbers
	DY64	DY66	DY67	DY68	Sum	Percentage of Sum Total
NeuN/DARPP-32	199	307	242	124	872	31.39
NeuN/Calb	71	3	0	88	162	5.83
NeuN/DARPP-32/Calb	490	374	503	377	1744	62.78

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Figure 12. — Distributions of NeuN/DARPP-32 cells, NeuN/calbindin cells and NeuN/DARPP-32/calbindin cells in the human and rat striosomes and matrix. (a) Bar chart comparing the proportions of NeuN cells colabelled with DARPP-32, calbindin, or a combination of DARPP-32 and calbindin in the dorsal human striosomes and matrix. The chart shows a higher proportion of NeuN/DARPP-32 and NeuN/DARPP-32/calbindin cells in the striosomes. By contrast, a higher proportion of NeuN/calbindin cells exists in the matrix in comparison to striosomes. (b) Considering only the NeuN-positive cells that were also labelled with at least one SPN marker, and discounting the NeuN only cell population in the dorsal human striosomes, 38% of SPNs or SPN-like cells were labelled with DARPP-32, whereas 42.6% were labelled with calbindin, and 19% are labelled with both DARPP-32 and calbindin. (c) Considering only the NeuN-positive cells that were also labelled with at least one SPN marker, and discounting the NeuN only cell population in the dorsal human matrix, 24% of SPNs or SPN-like cells were labelled with DARPP-32, whereas a large proportion of cells were labelled with calbindin (61.6%), and 13.9% were labeled with both DARPP-32 and calbindin. (d) A bar chart comparing the proportions of NeuN cells colabelled with DARPP-32, calbindin, or a combination of DARPP-32 and calbindin in the rat striosomes and matrix. A large proportion of the cells in the matrix exhibited NeuN/DARPP-32, but in the matrix, most of the cells were colabelled for NeuN, DARPP-32, and calbindin. (e) Considering only the NeuN-positive cells that were also labelled with at least one SPN marker, and discounting the NeuN only cell population in the rat striosomes, 89.7% of SPNs or SPN-like cells were labelled with DARPP-32, whereas 1% of the SPNs or SPN-like cells were labelled with calbindin only. A small portion (9%) were labelled with both DARPP-32 and calbindin. (f) Considering only the NeuN-positive cells that were also labelled with at least one SPN marker, and discounting the NeuN only cell population in the rat matrix, 31.4% of SPNs or SPN-like cells were labelled with DARPP-32. With respect to calbindin only SPNs or SPN-like cells, a small portion (5.8%) were immunolabelled with calbindin, but over half of the SPN or SPN-like cells (62.8%) were labelled with both DARPP-32 and calbindin.

Restricting our analysis of the human striatal regions examined to the cells labelled with SPN markers (DARPP-32 and calbindin, excluding the NeuN only cell population), we found that DARPP-32 positive only cells constituted 38.4% of SPNs or SPN-like cells in the striosomes. Calbindin positive only cells constituted 42.6% of SPNs or SPN-like cells in the striosomes. Cells that were DARPP-32 and calbindin positive made up 19% of SPNS or SPN-like cells in the striosomes (Figure 12b). In the dorsal human matrix compartments (n = 4, 2 sections per case), DARPP-32 positive only cell constituted 24.6% of SPNs or SPN-like cells. Calbindin positive only cells constituted 61.6% of SPNs of SPN-like cells, and cells that were DARPP-32 and calbindin positive made up 13.9% of SPN-like cells in the matrix compartments (Figure 12c). In a complementary analysis, we found that DARPP-32 immunostaining striosomes was present in 57.4% of all SPN or SPN-like cells in the striosomes, and was present in 38.5% of all SPN or SPN-like cells in the matrix.

In the rat striatum, we found that the NeuN/DARPP-32 population by proportion was higher in the rat striosomes than in the matrix. However, the NeuN/DARPP-32/calbindin cell population was higher in the matrix when compared to the same cell population in the striosomes (Table 3, Figure 12d). For cells labelled with SPN markers (DARPP-32 and calbindin, excluding the NeuN only cell population) DARPP-32 positive only cells constituted 89.7% of SPNs or SPN-like cells in the striosomes. Calbindin-positive cells constituted a mere 1% of all SPNs or SPN-like cells in the striosomes, and cells that were DARPP-32 and calbindin positive comprised 9% of SPNs or SPN-like cells in the rat striosomes (Figure 12e). These proportions were indeed different in the rat matrix, with DARPP-32 positive only cells comprising 31.4% of SPNs or SPN-like cells in the matrix; however calbindin positive only cells constituted 5.8% of the SPNs or SPN-like cells. Cells that were DARPP-32 and calbindin positive represented 62.8% of SPNs or SPN-like cells in the rat matrix (Figure 12f). DARPP-32 was present in almost 99% of all SPN or SPN-like cells in the rat striosomes, and DARPP-32 was also present in most such cells in the matrix (94% of all SPN or SPN-like cells in the rat matrix). Thus, and to summarize, one could contextualize these results to mean that the distribution of DARPP-32 across the rat striatum, taking into consideration the striosome and matrix compartments, is relatively homogenous.

3.11. DARPP-32 in the globus pallidus of post-mortem human brain

A qualitative examination of DARPP-32 immunoreactivity in the globus pallidus indicated that DARPP-32 was also present in the woolly fibres of both the external and internal segments of the globus pallidus (Figure 13). DARPP-32 immunoreactivity appeared to be homogenously distributed throughout both segments of the globus pallidus (Figure 13b, c).

Figure 13. — DARPP-32 is also present in the fibres of the external globus pallidus and the internal globus pallidus. (a) A photomacrograph of a globus pallidus section from a normal brain case (H160). (b and c) Photomicrographs of GPe (b) and GPi (c) indicate that DARPP-32 is present in the woolly fibres of the external and internal segments of the post-mortemhuman globus pallidus. GPe, globus pallidus externa; GPi, globus pallidus interna. Scale bars: 0.5 cm (a), 100 μm (b and c)

4. DISCUSSION

Here we demonstrate that in the human brain, the striosomal system of the dorsal striatum is marked by its strong expression of DARPP-32 immunostaining, relative to that of the surrounding matrix, and that an as yet further identified population of widely but sparsely distributed cells in the matrix compartment also exhibit strong DARPP-32 immunoreactivity. The strength of these findings rested on the reliability of the antibodies that we used. We tested the selectivity of the antibodies by Western blotting, immunoprecipitation and mass spectrometry, and we made comparisons of the findings with multiple DARPP-32 antibodies to test our use of the mouse monoclonal, rabbit monoclonal and goat polyclonal DARPP-32 antibodies (respectively, from Creative Diagnostics, Cell Signaling and Santa Cruz). Previous studies in rodents and rhesus macaque monkeys have suggested heterogeneity of DARPP-32 immunostaining in the striatum (Agnati et al., 1988; Ouimet, Lamantia, Goldman-Rakic, Rakic, & Greengard, 1992; Ouimet et al., 1984), without identifying different regions as being related to the striosome-matrix divisions of the striatum. Studies of the human striatum have also reported on DARPP-32 immunostaining, but without mentioning compartmental distributions (Guo et al., 2012; Hayakawa et al., 2013). Here, in most brain samples, we were able to take advantage of special post-mortem processing incorporating intravascular perfusion fixation of the brain tissue. We found clear definition of DARPP-32-positive striosomes as identified by the known negative striosome marker, calbindin, in the caudate nucleus with less prominent, but recognizable, compartmentally differentiated zones.

The striosome-predominant DARPP-32 expression patterns that we delineate here in dorsal striatum of the post-mortem human brain could have major implications for understanding the signalling properties of striatal cells, potentially including subclasses of SPNs in the human striatum. These results could, in turn, provide an improved basis for understanding disease states that affect the human striatum, such as Huntington’s disease and X-linked dystonia parkinsonism.

4.1. Regional distribution of DARPP-32

Our findings add DARPP-32 to a rich inventory of neurotransmitter-related molecules that have different patterns of expression in striosomes and matrix. For the human striatum, there are other neurochemicals that have been shown to favour either the striosome or matrix compartment such as acetylcholinesterase (Graybiel & Ragsdale, 1978), enkephalin (Emson, Arregui, Clement-Jones, Sandberg, & Rossor, 1980) and calbindin (Ferrante et al., 1991; Holt et al., 1997). There appears to be limited mention in the literature of the compartmental organisation of DARPP-32-positive cells into a specific striosomal/matrix pattern in the striatum of adult humans (Khan et al., 2021). DARPP-32 is a protein that relies heavily on dopamine for its functions as a phosphatase and kinase inhibitor. Furthermore, the basal ganglia nuclei are dependent on dopamine for optimal neurological functioning. Based on the increased presence of DARPP-32-positive SPNs in striosomes, compared to the matrix, we suggest that striosomes of the human striatum, especially those of the caudate nucleus, receive substantial dopamine-containing input.

4.2. Species difference with respect regional organization of striosomes and matrix

One previous immunohistochemical study has noted the patchy organisation of DARPP-32 immunoreactivity in the primate striatum (Ouimet et al., 1992). By contrast, there are multiple rodent-centric publications in which DARPP-32 immunostaining was found to display a reasonably homogeneous distribution throughout the striatum. Hallmark immunohistochemical studies of DARPP-32 in the rat striatum (Anderson & Reiner, 1991; Ouimet, Langley-Gullion, & Greengard, 1998; Ouimet et al., 1984) state that DARPP-32 is expressed in over 90% of medium-sized cells within the rat striatum, and propose that thus, DARPP-32 is validated to be used as an identifier of striatal projection neurons, or SPNs. The publications suggest that DARPP-32 identifies an almost complete majority of SPNs in the rat striatum, and DARPP-32 shows a relatively homogenous distribution across cell bodies and neuropil of the rat striatum. Our qualitative and quantitative investigations within this current publication confirm and corroborate this evidence for the rat. These findings laid way for other studies to build further striatal anatomical knowledge, with the widely held assumption that DARPP-32 identifies a large majority of SPNs in the rat striatum and the human striatum (Langley, Bergson, Greengard, & Ouimet, 1997). Only one rat study, to our knowledge, has alluded to potential hetereogeneity of DARPP-32 distribution in the rat striatum (Agnati et al., 1988), in contrast to the majority of rodent studies.

At the start of this study, we anticipated that the vast majority of SPNs or SPN-like cells in the human striatum would be labelled with DARPP-32. Our evidence based on examination of both neuropil and cell body immunostaining clearly has indicated that DARPP-32 striosome-matrix heterogeneity is present in the human striatum, that this is most pronounced in the dorsal caudate nucleus and adjoining anterodorsal part of the putamen, and that the DARPP-32 distributions are striosome-predominant. Yet we did find many DARPP-32 immunostained cells scattered throughout the matrix compartment. These results suggest that a marked species difference exists between the neurochemistry of the rodent and human striatum, or that different isoforms are represented in our analysis. It has been suggested that there could be some DARPP-32 heterogeneity in the rodent striatum (Agnati et al., 1988; Ouimet et al., 1984) and in the primate striatum (Ouimet et al., 1992), the presence or absence of DARPP-32 specifically in striosomes has not been indicated. In the present study, it appears that the staining pattern of the human is different to that in the rat, as our studies in the rat show no marked presence or notable striosome enhancement of DARPP-32 expression in striosomes in the rat striatum. This distribution of DARPP-32 within the striosomes and matrix appears to be a distinguishing feature unique to the mature human striatum (Khan et al., 2021).

Moreover, while some previously investigated neurochemicals tend to identify either the striosome (dopamine receptor D1) or matrix (calbindin) compartments consistently across the rodent and human striatum (Besson, Graybiel, & Nastuk, 1988; Crittenden & Graybiel, 2011; Gerfen et al., 1985; Holt et al., 1997; Liu & Graybiel, 1992; Murrin & Zeng, 1989), there appears to be conflicting evidence and conclusions regarding the distribution of TH within the rodent and human striatum. Immunohistochemical localization studies of TH within the rat brain constantly detail the presence of TH within the fibres and axon terminals of the entire rat striatum, but make no mention of the organization of TH-positive fibres into a striosome/matrix pattern organization (Hökfelt, Johansson, Fuxe, Goldstein, & Park, 1977; Pickel, Beckley, Joh, & Reis, 1981; Tashiro et al., 1989). Thus, from these publications, the reader would assume that TH is homogenously distributed through the rodent striatum. However, there is published immunohistochemical evidence that in the human striatum, TH is enriched in the matrix relative to striosomes, whereas the dopamine transporter, DAT, is enriched in the striosomes at least in non-human primate (Graybiel, Hirsch, & Agid, 1987; Moratalla et al., 1992). Thus, DARPP-32 is not the only molecule that is not synonymously distributed in the same manner across both rodent and human striatum, and interestingly, DARPP-32 and TH both belong to the wider dopamine system. Further investigation is highly warranted in this regarding these components of the dopamine system.

4.3. Striosome architecture and DARPP-32 striatal cell density in striosomes versus matrix

Previous suggestions indicate that striosomes in posterior striatal regions show more homogeneity than striosomes located more anteriorly (Bernácer, Prensa, & Giménez-Amaya, 2008). For the multi-labelled experiments that we performed, the anterior to the anterior commissure were examined, giving us a view of DARPP-32-, calbindin- and enkephalin-stained sections in the dorsal and anterior part of caudate nucleus and the dorsomedial putamen. Thus, our findings in regard to striosomal architecture and striosome border identification are limited to these relatively rostral regions.

This work demonstrates intense DARPP-32 staining corresponding to calbindin-negative regions, and the central part of the regions seen as enkephalin-positive rings around enkephalin-poor striosomes. Furthermore, striosomes typically consist of an enkephalin-rich ring surrounding an enkephalin-poor/calbindin-poor centre, and this is particularly seen in our staining of a striosome from the dorsal human putamen, confirming the finding of Prensa and colleagues (1999) and others showed that the striosome compartment is in itself heterogenous as seen in cross-section, consisting of a ‘core’ and a peripheral zone (Chesselet & Graybiel, 1983; Graybiel, Ragsdale, Yoneoka, & Elde, 1981; Graybiel & Ragsdale, 1978). In post-mortem human tissue, the absence of calbindin in striosomal areas indicated the ‘core’ of striosomes (Prensa et al., 1999), whereas high enkephalin immunoreactivity typically corresponds to the entire striosome (Prensa et al., 1999; Waldvogel & Faull, 1993). These observations are congruent with our own findings in striosomes for the post-mortem human dorsal striatum.

Furthermore, our study further supports previous evidence of two zones within striosomes that display different neurochemical properties, that suggests that the two zones hold different functional roles within the striosome compartment (Brimblecombe & Cragg, 2017). In particular, the higher presence of enkephalin in the periphery is postulated to be a source of enkephalinergic striatopallidal fibres that terminate in the globus pallidus externa (Prensa et al., 1999). This suggests that these two zones may belong to different anatomical systems.

Stereotaxic tracing studies in the primate suggest that the lateral part of the horizontal band of the substantia nigra pars compacta projects strongly to striosomes (Langer & Graybiel, 1989). It has also been observed that nigrostriatal dopaminergic innervation is mainly directed towards the core of the striosomes (Prensa et al., 1999). The striosomes in the Prensa (1999) study also showed positive acetycholinesterase (AChE) immunoreactivity within the core.

DARPP-32-positive cells were also present within the matrix portions of the caudate nucleus and putamen, although not at the same density. Indeed, our results on the human striatum indicate significantly different DARPP-32-positive cell densities in striosomes when compared to the matrix. Likewise, AChE immunostaining exhibits moderate intensity within the matrix, which is suggestive of different nigral cell populations innervating different parts of the striatum through different dopaminergic pathways (Prensa et al., 1999). Key findings from primate and rat studies obtained with classical methods include the partition of dopaminergic midbrain neuronal subpopulations into those that project to the matrix and others that project to striosomes (Langer & Graybiel, 1989; Prensa & Parent, 2001). The functional implications of these findings are that cortical and thalamic projections to the striosome and matrix compartments are under differing dopaminergic modulation (Doig, Moss, & Bolam, 2010; Huerta-Ocampo, Mena-Segovia, & Bolam, 2014; Langer & Graybiel, 1989; Martel & Galvan, 2022). Contextually, we suggest the possibility that the nigrostriatal dopaminergic innervation may be separated anatomically within the substantia nigra itself, depending on whether the striosomal core, the striosomal peripheral zone, or the matrix is innervated by dopamine. This organization would permit local dopamine modulation of corticostriatal or thalamostriatal afferents in the striosomal core, the striosomal peripheral zone, or the matrix.

Given that the lateral part of the horizontal band of the substantia nigra pars compacta projects strongly to striosomes (Langer & Graybiel, 1989) and DARPP-32 is highly concentrated in dopamine-recipient neurons (Svenningsson et al., 2004), one possibility, yet to be tested, is that, as DARPP-32-positive cells are present in high density in the striosomes, and at a moderate density in the surrounding matrix, DARPP-32 is a marker of striatal cells receiving dopaminergic innervation from the deep tier and perhaps more lateral substantia nigra pars compacta.

4.4. In the human striatum examined post-mortem, DARPP-32-positive striosomes are more distinct in the caudate nucleus than in the putamen

Within the striatal sections from the human brains, we found that the DARPP-32-positive striosomes in the dorsal caudate nucleus were more distinct, were more pronounced, and had more defined borders than those in the putamen, except for that part of the dorsomedial putamen closest to the caudate nucleus. These findings were consistent throughout a rostral-to-caudal gradient, when assessing the striatum at the level of the head of the caudate nucleus, anterior to the level of the anterior commissure, at the level of the anterior commissure, and at the level of the globus pallidus. Our findings with regards to the distinctiveness of striosomes in the caudate nucleus, as opposed to much of the putamen, are harmonious with previous reports in humans and primates (Holt et al., 1997; Langer & Graybiel, 1989; Roberts & Knickman, 2002). The publication by Roberts and Knickman has stated that caudate nucleus striosomes exhibited higher calbindin synaptic density than striosomes in the putamen. This potentially indicates that, due to the higher density of synapses received by striosomes in the caudate nucleus, these could be more robust and sharply defined. Previous findings from Holt et al. (1997) in post-mortem human tissue indicate that enkephalin-rich regions (striosomes) of the dorsal caudate nucleus are more distinct and exhibit more immunoreactivity than enkephalin-rich striosomes in the dorsal putamen. Similar findings are seen with human striatal tissue immunostained for choline acetyltransferase (ChAT), AChE and substance P (Holt et al., 1997), in which striosomes in the putamen were consistently observed to have diffuse borders and were not sharply defined, unlike striosomes of the caudate nucleus (Holt et al., 1997). Our observations of striosomal DARRP-32 expression are thus congruent with previous observations of striosomal anatomy in the human, further strengthening our proposition that DARPP-32 identifies striosomes in the human striatum.

4.5. The absence of detectable DARPP-32 immunostaining in identified striatal interneuronal populations of the human striatum

With immunofluorescent techniques, we did not find co-localisation of DARPP-32-positive striatal cells with the parvalbumin-, calretinin- or NPY-positive, presumed interneuronal subtypes. The general morphology of the DARPP-32-positive neurons identified in our study was not consistent with observed and previously noted morphology of interneurons (Cicchetti et al., 2000). As the majority of previous colocalisation studies regarding the striatal distribution of DARPP-32 and interneuronal markers have been conducted in rats and non-human species, it will be necessary to examine these new human-centric anatomical findings within the context of previous knowledge. Consonant with our findings, in a previous study with rats, DARPP-32 demonstrated a high level of colocalisation with striatonigral projection neurons (identified by substance P) and also with striatopallidal neurons (identified by enkephalin), but no DARPP-32/ChAT or DARPP-32/NPY colocalisation was apparent in interneurons (Anderson & Reiner, 1991). In a study of the rat striatum, DARPP-32 (used as a pan SPN marker) also did not yield evidence colocalization with any striatal cell populations positive for calretinin (Figueredo-Cardenas, Medina, & Reiner, 1996). Even in investigations of DARPP-32 immunoreactivity in the pigeon striatum demonstrated that DARPP-32 was widely prevalent in SPNs, but was largely absent from cholinergic (ChAT-positive) and NPY-positive interneurons (Reiner, Perera, Paullus, & Medina, 1998). Thus, the general literature consistently reports that DARPP-32 is found in SPNs, and also consistently reports a lack of DARPP-32 presence in striatal interneurons. Our results are consistent with this view, suggesting that the exclusivity of DARPP-32 in a select striatal cell population is conserved across different species.

4.6. DARPP-32 versus calbindin as a potential SPN marker

Our study demonstrates that DARPP-32-positive cell bodies match the Type 1 Golgi neurons identified by Graveland et al. (1985), which are typically categorised as SPNs. These characteristics included cell soma diameter, dendritic branching pattern, and representation of spines on dendrites, which were again similar to previous morphological definitions of SPNs (Braak & Braak, 1982). To date, there have been no studies in post-mortem human brain tissue evaluating the use of DARPP-32 as an SPN marker in comparison to calbindin, nor studies documenting potential colocalization of DARPP-32 and calbindin. However, we have observed additional putative SPN subtypes in the human.

4.7. Different cell-type proportions in striosomes and matrix, with consideration of the human and rat striatum

Previous studies have indicated that in the rat striatum, up to 96.4% of the SPNs expressed DARPP-32 (Ouimet et al., 1998). Our qualitative observations in the human do not fully conform to this pattern. Double immunofluorescence studies for calbindin and DARPP-32 led to the conclusion that there are three different SPN or SPN-like cell populations showing these markers within the normal human dorsal striatum. The first cell population was identified as DARPP-32 only positive; the second population was calbindin only positive, and the third population showed colocalization for both DARPP-32 and calbindin. These three populations of SPNs or SPN-like cells exist in the striosome compartment as well as in the matrix, but the specific proportions differ in each neurochemical compartment. Striosomes hold a concentrated number of DARPP-32-positive neurons, but DARPP-32-positive cells are distributed throughout the matrix, albeit somewhat less densely. Furthermore, similar to findings for the rat, we observed that DARPP-32 also labels the axons and dendrites that presumably arise from DARPP-32-positive cell soma. Together, these findings may support the concept of different dopamine processing circuits within the striosome and matrix compartments in the human striatum.

The origin of the neuropil labelling that so clearly distinguishes striosomes in the caudate nucleus of the human striatum remains to be identified. Previous evidence suggests, however, that both the nigrostriatal innervation and the striatonigral innervation reflect striosome-matrix compartmentalization, and markers of the dopamine system confirm this for the human brain (Gerfen, Baimbridge, & Thibault, 1987; Graybiel et al., 1987; Holt et al., 1997). These findings can also be discussed in the context of striosomal SPNs and their ability to form close associations with bundled dendrites of ventral tier neurons in striosome-dendron bouquets and the so-called posterior cell cluster of the substantia nigra pars compacta (Crittenden et al., 2016). It has not yet been possible to trace these circuits in the human nigrostriatal system, but snRNA-seq methods indicate heterogeneity (Matsushima et al., 2023). Our findings raise the possibility that different types of DARPP-32-positive SPNs could also have different afferent and efferent connections in the human brain, thus strengthening the implication that the two compartments are segregated in terms of processing circuits, especially those modulated by DARPP-32 (Gerfen et al., 1985; Herkenham, Edley, & Stuart, 1984; Penny, Wilson, & Kitai, 1988).

Striosomes in the rostral striatal regions that we studied receive inputs from the prefrontal/orbitofrontal cortex and likely other association areas are thought to be responsible for or related to limbic processing (Amemori et al., 2021; Eblen & Graybiel, 1995; Everitt, Dickinson, & Robbins, 2001). The matrix compartment, by contrast, receives projections from the sensory-motor and associative cortices and is potentially responsible for mechanisms related to sensorimotor processing (Crittenden & Graybiel, 2011; Kincaid & Wilson, 1996; Parent & Hazrati, 1995; Parthasarathy & Graybiel, 1997). The strong expression of DARPP-32 in the neuropil of striosomes could suggest that the heightened expression of DARPP-32 in the striosomes of the caudate nucleus, especially, could in particular influence the powerful prefronto-striosomal circuit terminating in the striosome-dendron bouquets of the substantia nigra (Crittenden et al., 2016).

Our findings in the rat striatum indicate that while the striosomes hold a large population of SPNs or SPN-like cells that are almost completely DARPP-32 positive, a large majority of cells in the matrix also contain DARPP-32 (~94%). As our study confirms previous findings of DARPP-32 immunoreactivity in the rodent striatum, it is thus understandable why many previous studies have preferred the use of DARPP-32 over calbindin in the rodent striatum as a pan-SPN marker (Anderson & Reiner, 1991; Ouimet et al., 1998). Previous literature has suggested that in the rat, the DARPP-32 signalling cascade plays a significant role in the entire caudatoputamen projection system to the globus pallidus, entopeduncular nucleus and the substantia nigra, which is consonant with the presence of DARPP-32 in a majority of the SPNs (Ouimet et al., 1998). Although D1 receptors are only found in around 59% of all DARPP-32 positive SPNs in the rat striatum, it is important to remember that there are other signal transduction pathways that could regulate DARPP-32, reminding us that DARPP-32 is not solely dependent on D1 receptor activation for its own regulation (Langley et al., 1997). Altogether, it is suggested that regardless of the signalling transduction pathways for DARPP-32, and as DARPP-32 is present in almost all the striatal projection neurons in the rat, the DARPP-32 cascade could represent one common pathway for convergent afferents to modulate striatal projections to outflow nuclei (Ouimet et al., 1998).

Thus, these species-specific differences in the distribution of DARPP-32 raise the question of differences in signalling transduction pathways, basal ganglia circuitry, and anatomy that exists between the striatum of the rat and human, albeit taking into account our concentration on the anterior caudate nucleus and putamen in the human brain material. It is apparent that a significant amount of further work needs to be done to fully understand these differences and to account for them when constructing rodent models for various neurological conditions, in the future.

4.8. Pallidal DARPP-32 expression in post-mortem human tissue

We observed strong DARPP-32 labelling of both segments of the post-mortem human globus pallidus, confirming previous work in multiple species (Hemmings Jr, Walaas, Ouimet, & Greengard, 1987; Ouimet & Greengard, 1990; Ouimet et al., 1992; Sen, Parishar, Pundir, Reiner, & Iyengar, 2019). Ouimet et al. in the primate brain (1992) reported that the pallidum is filled with DARPP-32-immunoreactive puncta that are aligned along dendrites. As it is understood that the striatum (caudate nucleus and putamen) sends projections to the globus pallidus (Jahanshahi, Obeso, Rothwell, & Obeso, 2015; Lévesque, Bédard, Cossette, & Parent, 2003; Parent, 1990; Parent & Hazrati, 1995; Wilson, 1914), the authors interpret that the DARPP-32-positive puncta in the primate and rodent globus pallidus (presumed analogue of the external pallidal segment of primates) represent nerve terminals that project from DARPP-32-positive cells in the striatum (Hemmings Jr et al., 1987; Ouimet et al., 1992). We were unable to confirm this finding, but we did observe typical ‘woolly fibre’ patterns in both pallidal segments. Given that DARPP-32 phosphorylation and resultant activity is dependent upon dopamine (which operates as a first messenger), the presence of DARPP-32 in the striatum and globus pallidus, both dopaminoceptive basal ganglia regions, is likely essential for dopamine-modulated synaptic interactions within the larger basal ganglia circuitry.

4.9. Conclusions

Our findings suggest that in the human brain, DARPP-32 may hold special relevance for the functions of striosomes, especially in the dorsal and anterior caudate nucleus and neighbouring dorsomedial putamen. We did not observe such distinct compartmental differentiation in rat, applying the same antibodies for detection. It is possible that species specificities interfered with DARPP-32 labelling in the rodents, or that the exaggerated DARPP-32 labelling of striosomes was due to some cross-over antigenicity. But multiple tests of the selectivity of the antibodies used suggested that this was not so. We found remarkably widely scattered DARPP-32 in the human matrix at the levels studied, but without such heavy labelling of neuropil as was present in the striosomes. In fact, in there was a greater density of DARPP-32-positive neurons in striosomes than in the matrix in the human material examined; but it was not clear whether or not they could have accounted for the dense neuropil labelling of the striosomes. These are major unknowns that hinder comprehensive interpretation of the staining patterns, but they do suggest that both compartmentalized neuropil and a dispersed, widely distributed set of cells in the matrix contribute to the sphere of influence of DARPP-32 in the human striatum.

We found that a subset of the SPNs or SPN-like cells identified as DARPP-32-positive were co-labelled for calbindin. The DARPP-32-positive cells were less numerous than the calbindin-positive cells, and the compartmental distributions were uneven, as the DARPP-32-positive neurons were more numerous in the calbindin-poor striosomes than in the matrix. We were unable to compare the distributions of DARPP-32 to other calcium binding-positive SPNs, of particular interest being CalDAG-GEFI, a calcium binding protein that targets a ras-family guanine nucleotide factor (Kawasaki et al., 1998), which is differentially expressed in striosomes in the rodent and strongly down-regulated in rats exhibiting l-DOPA-induced dyskinesias (Crittenden et al., 2009).

DARPP-32 activity is dependent on activation by dopamine D1 and D2 receptors. Depending on which receptor subtype is activated, DARPP-32 acts to inhibit either PP1 or PKA (Fienberg et al., 1998). D1 receptor activation and the resultant downstream effects modulating calcium currents in striatal neurons have been studied (Fienberg et al., 1998; Floran, Aceves, Sierra, & Martinez-Fong, 1990). The application of D1 receptor agonists in rat striatal slices decreases excitatory postsynaptic potentials (Cepeda, Buchwald, & Levine, 1993). Furthermore, D1 receptor agonists have been shown to influence the Ca²⁺-dependent release of GABA by SPNs in striatal sections from rat brains (Floran et al., 1990). Whole cell voltage clamp studies conducted in neurons from wild-type and DARPP-32 knockout mice showed that Ca²⁺ currents were attenuated by around 50% in striatal neurons in the knockout mice when D1 receptor agonists were applied (Fienberg et al., 1998). Yet to be tested is whether calbindin and DARPP-32 could be special regulators of the striosomal system, with the dispersed DARPP-32 cells of the human striatum participating in other regulation. Nevertheless, the identification here of neurons positive for DARPP-32-only, calbindin-only, and both DARPP-32 and calbindin suggest that these considerations likely apply differently to different SPN or SPN-like populations. Our findings lay the groundwork for further study of these potentially functionally differentiated circuits in the human brain.

Key Points.

DARPP-32 is highly concentrated in cells and neuropil of striosomes in post-mortem human brain tissue, particularly in the dorsal caudate nucleus.
Scattered DARPP-32-positive cells are found in the human striatal matrix.
Calbindin and DARPP-32 do not colocalize within every spiny projection neuron in the dorsal human caudate nucleus.

Acknowledgements

We extend our thanks to the families who have generously donated brain tissue to The Neurological Foundation Human Brain Bank in the Centre for Brain Research. The authors wish to thank M. Eszes (Human Brain Bank), K. Hubbard, and C. Lill (Research Technicians) for their continuous hard work and assistance. The authors wish to thank C. Wuethrich for his technical assistance towards this publication. Our thanks to Dr. Lekha Jain for technical assistance with the immunoprecipitation techniques and advice associated with this project. The authors also wish to thank Martin Middleditch of the Mass Spectrometry Centre, The University of Auckland, Auckland, New Zealand for assistance with mass spectrometry analysis for various samples. Our thanks to Jessica Crockett for assistance with the acquisition of photomicrographs used in this publication. Further thanks to Jacqueline Ross and Richard Yulo from the Biomedical Imaging Research Unit at the Faculty of Medical and Health Sciences, The University of Auckland for their advice and assistance with confocal imaging and image management. Our thanks to Dr. Yasuo Kubota for proof-reading of the manuscript. This work was supported by the Health Research Council of New Zealand, the Maurice and Phyllis Paykel Trust, the Neurological Foundation of New Zealand, the Collaborative Center for X-linked Dystonia Parkinsonism, the National Institute of Mental Health, the Saks Kavanaugh Foundation, Robert Buxton, and Mr. James and Mrs. Joan Schattinger. C. J. Arasaratnam was supported by a University of Auckland Doctoral Scholarship.

Funding Information

HRC grant numbers 371-0464, 372-3033; MPPT grant 3712941, CCXDP grant 3719286, US National Institutes of Health grant R01 MH060379

Footnotes

Conflict of Interest

The authors declare no conflict of interest in the present study.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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PERMALINK

DARPP-32 Cells and Neuropil Define Striosomal System and Isolated Matrix Cells in Human Striatum

CJ Arasaratnam

JJ Song

T Yoshida

MA Curtis

AM Graybiel

RLM Faull

HJ Waldvogel

Abstract

Graphical Abstract

1. INTRODUCTION

2. MATERIALS AND METHODS

2.1. Brain tissue

Table 1:

2.2. Antibody characterization

Table 2:

2.3. Western blotting

2.4. Immunoprecipitation procedures

2.5. Immunohistochemical procedures

2.5.1. Immunoperoxidase labelling (select antibodies)

2.5.2. Immunoperoxidase labelling (Rabbit anti DARPP-32 only)

2.5.3. Immunofluorescence labelling

2.6. Imaging

2.6.1. Immunoperoxidase stained sections (selected antibodies)

2.6.2. Immunoperoxidase-stained sections (Rabbit anti DARPP-32 only)

2.6.3. Immunofluorescent-stained sections

2.7. Cell counts

2.7.1. Manual cell counts on immunoperoxidase-stained human striatal sections for calculations of density

2.7.2. Automatic cell counts on immunofluorescent stained post-mortem human and rat striatal sections

3. RESULTS.

3.1. Validation of the DARPP-32 antibodies with Western blotting techniques

Figure 1.

3.2. Mass spectrometry analysis of Western blot and immunoprecipitation samples

3.3. Consistency of immunostaining patterns and comparison of mouse and human striatal DARPP-32 immunostaining patterns

Figure 2.

3.4. DARPP-32 immunolabelling of neuropil in the dorsal human striatum was predominantly located in striosomes

Figure 3.

3.5. Striosome and matrix architecture in the normal human dorsal striatum: view at pre-anterior commissural levels with immunofluorescent staining for DARPP-32, calbindin and enkephalin, with comparisons to rodent patterns.

Figure 4.

Figure 5.

Figure 6.

3.6. In the human striatum, DARPP-32-positive striosomal borders are more distinct in the caudate nucleus than in most of the putamen except at rostral levels

Figure 7.

3.7. Immunodetectable DARPP-32 was localized in cells resembling SPNs in the caudate nucleus of the human striatum

Figure 8.

3.8. Higher density of DARPP-32-positive cell bodies in striosomes than the matrix of the dorsal human caudate nucleus

Figure 9.

3.9. Within the dorsal striatum of the human, DARPP-32 was not detectable in the striatal interneurons identified

Figure 10.

3.10. Double-immunofluorescent labelling of DARPP-32 and calbindin suggest the presence of three SPN or SPN-like cell populations in the dorsal striatum of the human and rat

Figure 11.

Table 3:

Figure 12.

3.11. DARPP-32 in the globus pallidus of post-mortem human brain

Figure 13.

4. DISCUSSION

4.1. Regional distribution of DARPP-32

4.2. Species difference with respect regional organization of striosomes and matrix

4.3. Striosome architecture and DARPP-32 striatal cell density in striosomes versus matrix

4.4. In the human striatum examined post-mortem, DARPP-32-positive striosomes are more distinct in the caudate nucleus than in the putamen

4.5. The absence of detectable DARPP-32 immunostaining in identified striatal interneuronal populations of the human striatum

4.6. DARPP-32 versus calbindin as a potential SPN marker

4.7. Different cell-type proportions in striosomes and matrix, with consideration of the human and rat striatum

4.8. Pallidal DARPP-32 expression in post-mortem human tissue

4.9. Conclusions

Key Points.

Acknowledgements

Funding Information

Footnotes

Data Availability Statement

References

Associated Data

Data Availability Statement

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