Quantitative immunocytochemistry of DARPP-32-expressing neurons in the rat caudatoputamen

doi:10.1016/S0006-8993(98)00724-0

Brain Research

Volume 808, Issue 1, 12 October 1998, Pages 8-12

https://doi.org/10.1016/S0006-8993(98)00724-0 Get rights and content

Abstract

DARPP-32, a dopamine and cAMP-regulated phosphoprotein with an apparent molecular weight of 32 kD, is enriched in dopaminoceptive brain regions. Chief among these regions is the caudatoputamen which contains a large number of DARPP-32-containing medium sized spiny neurons. Since medium spiny neurons are a heterogeneous population with respect to connections and chemical neuroanatomy, it seemed of interest to determine whether DARPP-32 is present in all medium-sized neurons, or only within a specific subpopulation. The present study used immunocytochemistry and quantitative analysis to address this issue. We demonstrate that DARPP-32 is contained in almost all medium-sized neurons (96.4%) and is not detected in large neurons. Taken together with previous observations that the DARPP-32-containing medium-sized neurons project heavily to all neostriatal targets, these data demonstrate that DARPP-32 is present in virtually all neostriatal output neurons. Thus, the DARPP-32 cascade represents a final common pathway through which convergent afferent fibers using a variety of neurotransmitter agents may modulate striatal outflow.

Introduction

DARPP-32 is a dopamine (DA) and cAMP-regulated phosphoprotein with an apparent molecular weight of 32 kD 14, 26, 36. Cyclic AMP-dependent protein kinase, activated by dopamine D₁ receptor stimulation, phosphorylates DARPP-32 [36]. Calcineurin, activated by D₂ receptor stimulation dephosphorylates DARPP-32 [19]. Phosphorylated DARPP-32 (but not dephosphorylated DARPP-32) inhibits protein phosphatase 1 (PP1) and thereby blocks the dephosphorylation of PP1 substrates [13]. Such substrates include the Na⁺/K⁺ ATPase 2, 7and voltage-regulated calcium channels [30].

DARPP-32 is enriched in brain regions that contain the dopamine D₁ receptor, an observation consistent with the concept that DARPP-32 phosphorylation is regulated by D₁ receptor activity 25, 37. The largest and best understood of the dopaminoceptive brain regions is the caudatoputamen (CP). A large number of medium spiny neurons in the CP are immunoreactive for DARPP-32 1, 25and biochemical studies have shown that the concentration of DARPP-32 in this region is very high [12].

Although the number of DARPP-32-containing neurons in the CP has been roughly estimated 1, 25it is not known whether biochemical pathways using DARPP-32 are present in all medium-sized neurons, or only within specific subpopulations. This issue is of interest because the CP is anatomically and neurochemically heterogeneous. The present study was undertaken to estimate the number of CP neurons that are engaged in DARPP-32-mediated signal transduction and to determine whether the DARPP-32-containing neurons are homogeneously distributed.

Section snippets

Materials and methods

Male Sprague–Dawley rats (300–500 gm) were deeply anesthetized with sodium pentothal (60 mg/kg) and transcardially perfused with 4% formaldehyde in sodium phosphate buffer (PB; 0.1 M, pH 7.4). After a 1 h. period of postfixation in situ, brains were removed and placed in fixative for an additional 18 h. Sections 30–50 μm thick were cut on a vibratome, collected in phosphate buffered saline (PBS; 0.01 M PB, 0.15 M NaCl, pH 7.5) and immunostained for DARPP-32 using a Vector Elite ABC kit (Vector

Results

The overwhelming majority of medium-sized neurons in the caudatoputamen were immunoreactive for DARPP-32 (Fig. 1). Of all medium-sized neurons counted, 96.4% expressed DARPP-32 (Table 1). In the remainder of the medium-sized neurons (3.6%), no immunoreactivity could be detected. The DARPP-32-negative medium-sized cells could not be distinguished from their DARPP-32-positive counterparts by morphological criteria. Both cell types had scant cytoplasm, round nuclei that stained weakly with cresyl

Discussion

The absence of DARPP-32 immunoreactivity from large interneurons suggests that the DARPP-32 cascade is not used to alter the physiology of these striatal local circuit neurons. Large neurons are believed to be GABAergic or cholinergic [3]and DARPP-32 is not detectable in neurons containing choline acetyl transferase [1]. The small population of medium-sized neurons in which DARPP-32 could not be detected suggests that these cells are not influenced by the DARPP-32 cascade. It cannot be assumed

Conclusion

In summary, DARPP-32 is present in virtually all of the CP projection neurons, and its state of phosphorylation is regulated by diverse neurotransmitters through their associated receptors. Taken together with the observation that DARPP-32 is present in virtually all striatal projection neurons, the data suggest that the DARPP-32 cascade represents one final common pathway through which convergent afferent fibers modulate striatal outflow.

Acknowledgements

This work was supported by NIH grant MH40899-11.

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Research reportQuantitative immunocytochemistry of DARPP-32-expressing neurons in the rat caudatoputamen

Abstract

Introduction

Section snippets

Materials and methods

Results

Discussion

Conclusion

Acknowledgements

Brain Res.

Brain Res.

Neuroscience

Brain Res.

Neuron

Exp. Neurol

Exp. Neurol.

Activation/deactivation of renal Na+,K+-ATPase: a final common pathway for regulation of natriuresis

FASEB J.

Characterization of cholinergic neurons in rat neostriatum. A combination of choline acetyltransferase immunocytochemistry, Golgi-impregnation and electron microscopy

Neuroscience

The precise localization of nigral afferents in the rat as determined by a retrograde tracing technique

Brain Res.

Parvalbumin-containing GABAergic interneurons in rat neostriatum

J. Comp. Neurol.

Ultrastructural features of immunoreactive somatostatin neurons in the rat caudate nucleus

J. Neurosci.

Na+,K+-ATPase in the choroid plexus. Regulation by serotonin/protein kinase C pathway

J. Biol. Chem.

The neostriatal mosaic: compartmental distribution of calcium binding protein and parvalbumin in the basal ganglia of the rat and monkey

Proc. Natl. Acad. Sci. U.S.A.

D1 and D2 Dopamine receptor-regulated gene expression of striatonigral and striatopallidal neurons

Science

Activation of NMDA receptors induces dephosphorylation of DARPP-32 in rat striatal slices

Nature

DARPP-32, a dopamine- and adenosine 3′:5′-monophosphate-regulated phosphoprotein: regional, tissue, and phylogenetic distribution

J. Neurosci.

DARPP-32, a dopamine-regulated neuronal phosphoprotein, is a potent inhibitor of protein phosphatase-1

Nature

Research report
Quantitative immunocytochemistry of DARPP-32-expressing neurons in the rat caudatoputamen

Activation/deactivation of renal Na⁺,K⁺-ATPase: a final common pathway for regulation of natriuresis

Na⁺,K⁺-ATPase in the choroid plexus. Regulation by serotonin/protein kinase C pathway

D₁ and D₂ Dopamine receptor-regulated gene expression of striatonigral and striatopallidal neurons