Altered Dopaminergic receptor binding sites in the caudate nucleus of subjects with Rett Syndrome. :::: Welcome to Child Trauma Academy :::::::::::

Altered Dopaminergic receptor binding sites in the caudate nucleus of subjects with Rett Syndrome.

Bruce D. Perry, MD., PhD
Domenico Vigilante
Dawna Armstrong MD

The ChildTrauma Academy
www.ChildTrauma.org

Formerly a partnership of:
Baylor College of Medicine
and Texas Children's Hospital

This is an Academy version of an abstract for the World Congress on Rett Syndrome.

Official Citation: Perry, BD, Vigilante, D. and Armstrong, D. Altered dopaminergic receptor binding sites
in the caudate nucleus of subjects with Rett Syndrome. Proceedings of the World Congress on
Rett Syndrome, Goteborg, Sweden, 1996

Introduction: One tenth of the profoundly retarded female population have Rett Syndrome (RS). The brain in RS, particularly the caudate nucleus, is decreased in size. Abnormalities of the biogenic amines have been hypothesized to be related to the pathogenesis of this disorder. Methods: The present study examined dopamine (D₁, D₂) receptor binding sites in subjects (4, 7, 7 & 31 yrs) who have been diagnosed with RS and three non-Rett brains (0.2, 5 & 11 yrs). The caudate nucleus was dissected from frozen brains and eight point saturation isotherms were performed using standard techniques. D₁ and D₂ -dopamine receptor binding sites were labeled using ³H-SCH 23390 (NEN DuPont 70 Ci/mmoles) and ³H-spiperone (NEN DuPont 18.5 Ci/mmoles) respectively. Results: Major findings include 1) lower D₂-receptor binding site density in subjects who had RS vs. non-RS (344.0 vs. 945.8 fmol/mg protein), while no differences were seen in the D₁ binding site density (272.8 vs. 242.8 fmol/mg protein). No group differences in radioligand affinity (Kd) were observed. Discussion: This finding suggests altered innervation of caudate by dopaminergic neurons. The decrease in D₂-binding sites (primarily pre-synaptic) in RS relative to non-RS suggests either altered innervation due to decreased dopaminergic innervation or inadequate neurotrophic support of these innervating dopaminergic neurons by caudate target sites. While no group differences in D₁binding site density were observed, the RS values are below published values in adolescents and adults (data not shown). The dramatic decrease in pre-synaptic sites and possible decrease in D₁binding site density (in comparison to published values) suggests altered dopaminergic neurotransmission in Rett Syndrome. Further studies examining neurodevelopmental aspects of dopaminergic neurotransmission in RS seem indicated. (Supported by the CIVITAS Initiative and NIH/NICDH: Rett Syndrome Research Program Project).

Background

Rett Syndrome (RS), a disorder of unknown etiology in girls, manifests as a severe mental and motor deficiency during the first years of life. Several lines of research have suggested that altered dopaminergic neurotransmission plays a role in the pathophysiology of RS. Specifically, some features the motor deficit resemble Parkinson's disease (Fitzgerald et al ‘90); melanin in some of the large neurons of the substantia nigra is reduced (Seitelberger and Jellinger '86 and Kitt et al ‘90 ) and bromocriptine has alleviated some of the symptoms in RS (Zappelia et al ‘86). These finding suggest a deficiency of dopamine in RS.

Studies which have been done on dopamine include: measurement of decreased amounts of dopamine in multiple brain sites in one case at autopsy (Riederer ‘85, Brucke ‘86), measurement of decreased amounts of dopamine in the brains (three Rett cases by Wenk et al ‘89, and 5 cases by Wenk et al ‘91) normal D₁ receptors and decreased dopamine reuptake in the caudate and putamen in 11 cases of RS (Wenk ‘95), decreased D₂ receptor binding in the putamen (Riederer et al ‘86) decreased D₂ receptors as defined with PET (Naidu et al ‘92) and increased D₂ in the putamen as defined with SPECT (Chiron ‘93) (Table 1 and 2). Studies of homovanillic acid (HVG) in the CSF of patients with RS have been conflicting (Zoghbi et al ‘86, ‘87, Perry et al ‘88 and Lekman et al ‘90). There may be a decrease of HVG in the CSF in the youngest Rett patients (Percy ‘92). These studies do not readily clarify the role of dopamine in RS, warranting further study. In this study abnormal amounts of D₂ receptor binding were identified in the caudate nucleus in four Rett brains.

Subjects	Age	Gender	Cause of Death
Rett 1	4 years	f	sudden death
Rett 2	7 years	f	sudden death
Rett 3	8 years	f	gastric perforation and sepsis
Rett 4	4 years	f	status epilepticus
Non Rett 1 (normal brain)	2 month	f	congenital heart disease
Non Rett 2 (normal brain)	5 year	f	congenital heart disease
Non Rett 3 (normal brain)	11 year	f	neoplasm

Methods

Brain tissue was obtained from either Maclean Hospital Brain Tissue Resource, Texas Children's Hospital, or the Methodist Hospital.

Membrane Preparation for Brain Receptor Characterization: All regions are dissected and placed in an appropriate ice-cold TRIS wash buffer for processing. Wash buffers to be made up one day prior to dissection include 50 mM TRIS HCl buffer pH 7.4 @ 37 °c (w/ 50 mM NaCl and 5 mM EDTA ), (w/ 100 mM NaCl), (w/ 100 mM NaCl and 5 mM EDTA) and 50 mM TRIS HCl buffer pH 7.4 @ 25 °c w/ 100 mM NaCl for a -adrenergic, dopaminergic, and serotonergic receptor assays respectively. On the day of dissection all brain regions are homogenized in 8 mls of wash buffer for 5 minutes or until solubilized utilizing a CON-TORQUE homoginizer. These membrane solutions are then spun in a 4 °c refrigerated centrifuge at 50,000 xg for 15 minutes. Following the spin, the supernatant is decanted and discarded; the resulting pellets are frozen at -70 °c until resuspension with assay buffer on the day of the assay. Receptor radioligand binding methods for the a -2, 5-HT_1A, 5-HT₂, D₁ and D₂ are performed using adaptations from original technique and are described elsewhere. In brief,

a -2 adrenergic binding sites will be labeled using ³H-Rauwolscine (NEN DuPont, 80.5 Ci/mmole). Binding sites will be quantified using saturation studies (6 concentrations ranging from 0.1-5.0 nM). In brief, aliquots of tissue (final concentrations of 100-500 m g protein/ml) in 50 mM TRIS HCl assay buffer (pH 7.4, 37 °c w/ 50 mM NaCl) were incubated with increasing concentration of radioligand for 60 minutes at 25 °c. Parallel incubations in the presence of 0.1 mM Norepinephrine Bitartrate (RBI, Research Biochemical Inc.) will define specific binding. Final incubation volume was 500 m l.

5-HT1A binding sites will be labeled using ³H-DPAT (NEN DuPont, 143.80 Ci/mmole). Binding sites will be quantified using saturation studies (6 concentrations ranging from 0.1-5.0 nM). In brief, aliquots of tissue (final concentrations of 100-500 m g protein/ml) in 50 mM TRIS HCl assay buffer (pH 7.4, 37 °c w/ 120 mM NaCl) were incubated with increasing concentration of radioligand for 45 minutes at 25 °c. Parallel incubations in the presence of 1 mM 5-HT (Sigma) will define specific binding. Final incubation volume was 300 m l.

5-HT2 binding sites will be labeled using ³H-Spiperone (NEN DuPont, 18.5 Ci/mmole). Binding sites will be quantified using saturation studies (6 concentrations ranging from 0.2-5.0 nM). In brief, aliquots of tissue (final concentrations of 100-500 m g protein/ml) in 50 mM TRIS HCl assay buffer (pH 7.4, 37 °c w/ 120 mM NaCl) were incubated with increasing concentration of radioligand and 0.1 m M Haloperidol (McNEIL pharmaceutical injection; stock solution 5 mg/ml) for 60 minutes at 25 °c. Parallel incubations in the presence of 1.0 mM Mianserin (Sigma) will define specific binding. Final incubation volume was 520m l.

D₁ binding sites will be labeled using ³H-SCH 23390 (NEN DuPont, 70 Ci/mmole). Binding sites will be quantified using saturation studies (6 or 8 concentrations ranging from 0.05-3.0 nM). In brief, aliquots of tissue (final concentrations of 100-500 m g protein/ml) in 50 mM TRIS HCl assay buffer (pH 7.4, 37 °c w/ 120 mM NaCl AND 0.01% BSA) were incubated with increasing concentration of radioligand and 0.5 m M Ketanserin (RBI, Research Biochemical Inc.) for 60 minutes at 25 °c. Parallel incubations in the presence of 0.1 mM Fluphenazine (RBI, Research Biochemical Inc.) will define specific binding. Final incubation volume was 320 m l.

D₂ binding sites will be labeled using ³H-Spiperone (NEN DuPont, 18.5 Ci/mmole). Binding sites will be quantified using saturation studies (6 concentrations ranging from 0.5-10.0 nM). In brief, aliquots of tissue (final concentrations of 10-150 m g protein/ml) in 50 mM TRIS HCl assay buffer (pH 7.4, 37 °c w/ 120 mM NaCl AND 0.01% BSA) were incubated with increasing concentration of radioligand and 50 m M Mianserin for 45 minutes at 37 °c. Parallel incubations in the presence of 0.1 mM Haloperidol (McNEIL pharmaceutical injection; stock solution 5 mg/ml) will define specific binding. Final incubation volume was 520 m l.

For all assays, incubation is terminated by filtration under reduced pressure over Whatman GF/B filter paper using a Brandel Cell Harvester modified for radioligand binding assays. Reaction tubes are rinsed 3 times 5.0 mls with ice-cold 50 mM TRIS HCl buffer (pH 7.7 at 25 °c). For all ³H ligands, radioactive filters are placed in disposable glass mini-vials (Fisher), and 3.0 mls of CytoScint^TM ES* Scintillation Cocktail (ICN Biomedicals, Inc.) is added. The samples are allowed to equilibrate for at least 12 hrs. prior to being counted by liquid scintillation spectrometry (Packard 2500 TR) with efficiency of 40-50%

Protein Determination: Protein concentrations will be determined using a commercially available BCA protein assay kit (Pierce, Rockford, IL.), and read on a Beckman DU-60 spectrophotometer.

Data Analysis: Raw counts from all assays are transformed using RADLIG (1), a radioligand binding analysis package which contains EBDA and a version of LIGAND derived from the original (2). In all cases, saturation isotherms are best fit by a single site model and final derivation of radioligand affinity (Kd) and the density of binding sites (B_max) are made using weighted Eadie-Hofstee plots as described by Zivin and Waud (3) and adapted for LOTUS 1-2-3. Computer assistant analysis of binding isotherms indicate a single, saturable site and interaction. Specific binding varied as a function of the density of binding sites and concentration of radioligand (i.e. as the sites are saturated, specific binding decreases). Data is analyzed for statistical significance using 2-way ANOVA, and the criterion for significance was P<0.05.

Results

Major findings were found in the caudate nucleus and include a lower D₂-receptor binding site density in subjects who had RS vs. non-RS (344.0 vs. 945.8 fmol/mg protein), while no differences were seen in the D₁ binding site density (272.8 vs. 242.8 fmol/mg protein). No group differences in radioligand affinity (Kd) were observed (see Figure 1).

The analysis of all of the radioligand binding assays in each of the brain regions analyzed demonstrated no other group differences in density of binding sites or in affinity of radioligand.

Discussion

These results support the previous observations that dopaminergic innervation is altered in Rett Syndrome. Continued examination of this system in RS is indicated. Nomura and Segawa ‘84 suggested from their neurophysiologic studies of Rett girls that deficiency of a central brainstem monoamine system that normally projected widely and exerted diffuse activities might be responsible for the symptoms of Rett syndrome. However, many of the manifestations of RS are quite different from Parkinson’s' disease in which there is a dopamine deficiency, so that the pathoetiology must involve mechanisms other than deficiency of a functioning neurotransmitter. Because the symptoms of RS appear in the first year of life, before the brain has matured, deficiency of dopamine might affect the developing brain in a unique manner. It may deprive the brain of the necessary trophic influence of dopamine during a critical period of brain growth (Lipton et ‘90). Elucidation of the etiology of the decreased amount of dopamine may bring us closer to understanding the etiology of Rett Syndrome.