Neuropathological Study of the Dorsal Raphe Nuclei in Late-Life Depression and Alzheimer’s Disease With and Without Depression

Tissue samples were obtained from the Newcastle (U.K.) Brain Tissue Bank. All subjects were age 60 or older at the time of death. Full approval for this postmortem study was granted by the Newcastle and North Tyneside local research ethics committee, and consent for postmortem examination was obtained from each subject’s next of kin. Alzheimer’s disease subjects had been prospectively assessed in a longitudinal study of dementia and had met the criteria of the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association for probable Alzheimer’s disease (19) and had an Mini-Mental State Examination (MMSE) (20) score of >7 at initial assessment. A postmortem diagnosis of definite Alzheimer’s disease was made by using the criteria of the Consortium to Establish a Registry for Alzheimer’s Disease (21). The Alzheimer’s disease subjects had had annual research assessments, including assessment with the cognitive section of the Cambridge Mental Disorders of the Elderly Examination (22), and had been assessed every 6 months with the MMSE and the Cornell Scale for Depression in Dementia (23). Subjects who had scored >10 on the Cornell Scale for Depression in Dementia were assessed by a research psychiatrist to determine whether they met the DSM-III-R criteria for major depression or the DSM-IV criteria for major depressive disorder (ignoring the exclusion criterion of organic causation). Nondepressed Alzheimer’s disease subjects did not meet the DSM criteria for depression at any time point and had Cornell scale scores of <10. Depressed subjects had suffered at least one well-documented episode of DSM-III-R major depression or DSM-IV major depressive disorder, as determined by the treating clinician and by two independent research psychiatrists who reviewed the subjects’ case notes. Subjects were excluded if they had ever met the DSM-IV criteria for dementia, schizophrenia, or another severe mental illness. Subjects with cognitive impairment during depressive episodes were not excluded, provided the impairment was regarded clinically as secondary to the depressed mood and the postmortem examination of the brain showed no neuropathological evidence of changes consistent with dementia. We obtained information from the case notes on the subjects’ history of treatment with antidepressant medication and ECT. The comparison subjects had never had a depressive episode, showed no evidence of a dementing illness, and had been capable of independent living. Postmortem assessment of the comparison subjects also showed no evidence of known causes of dementia.

After the postmortem examination, the brainstem was fixed in a 10% buffered formalin solution and embedded in paraffin. The dorsal raphe nucleus was sampled at the level of the trochlear nucleus, and three sections were taken for analysis from each subject. Three series of sections, each consisting of a 6×10-μm section, a 2×20-μm section, and a 3×30-μm section, were cut. From each series, the seventh (20-μm) section was marked with Nissl stain to assist in identification of landmarks, and the adjacent sixth (10-μm) section was used for immunocytochemistry. Slides were coded so that all analyses could be carried out by evaluators who were blind to the subject’s diagnosis.

The dorsal raphe nucleus and its subnuclei were identified by using a standard brainstem atlas (24) and the cytoarchitectural study of the dorsal raphe nucleus by Baker et al. (25). The overall distribution of both Nissl-stained neurons and neurons that were immunoreactive to tryptophan hydroxylase (in tissue from comparison subjects and depressed subjects) corresponded well with these descriptions. The dorsal raphe nucleus was located in the central gray matter ventral to the cerebral aqueduct and fourth ventricle, with most of the nucleus dorsal to the medial longitudinal fasciculi. As previously described (25), the organization and arrangement of the individual subnuclei of the dorsal raphe nucleus at this level formed a fountain shape comprising a medial portion and two lateral wings extending along either side of the midline. Within the medial portion, two subnuclei were clearly visible. The interfascicular subnucleus was located between the medial longitudinal fasciculi, and the ventral subnucleus was immediately dorsal to the interfascicular subnucleus. Demarcation of these two subnuclei was aided by the apparent morphology of the interfascicular subnucleus neurons, which included a preponderance of elongated fusiform neurons that were aligned parallel with the midline. Within the lateral wings of the dorsal raphe nucleus, another two subnuclei were clearly visible: the ventrolateral subnucleus, which was located dorsolateral to the trochlear nucleus, and the dorsal subnucleus, which was immediately dorsal to the ventrolateral subnucleus and just ventral to the floor of the cerebral aqueduct. The ventrolateral subnucleus was easily identified because its densely packed neurons formed two prominent clusters on either side of the midline. The ventral border of the ventrolateral subnucleus was clearly separated from the trochlear nucleus by a narrow cell-free strip. In comparison, the neurons of the dorsal subnucleus were more scattered and occupied the largest volume of these subnuclei. The dorsal subnucleus was bilaterally organized but joined in the midline. Although neurons of the dorsal subnucleus merged with those of the ventrolateral subnucleus, the two subnuclei were clearly demarcated by their differing densities of neurons that were immunoreactive to tryptophan hydroxylase. As in previous studies (25), the dorsal subnucleus exhibited the lowest density of neurons, while the ventrolateral subnucleus had the highest density of neurons. These findings were evident in each of the three diagnostic groups (the comparison, depression, and Alzheimer’s disease groups).

Serotonergic cells were identified by staining for tryptophan hydroxylase, which is found only in serotonergic neurons (26). The extent of neurofibrillary pathology was quantified by staining for the microtubule-associated tau protein, the key component of neurofibrillary tangles in Alzheimer’s disease (27). Tissue sections were stained by using standard immunocytochemistry. After dewaxing in xylene and rehydration through graded alcohols, the sections were microwaved at 450 W for three 5-minute bursts in a 0.01M citrate buffer solution (pH 6) to optimize antigen retrieval (28). Endogenous peroxidase activity in the tissue was neutralized by washing the sections in a 3% solution of H₂O₂ in methanol for 15 minutes. After blocking with an appropriate serum, sections were incubated overnight at 4°C in a moist chamber with a monoclonal antibody to tryptophan hydroxylase (mouse IgG3 diluted to a ratio of 1:500) (Sigma-Aldrich Ltd., Dorset, U.K.). After the sections were washed, they were incubated for 30 minutes with an appropriate secondary antibody, then for 30 minutes with avidin-biotinylated horseradish peroxidase complex (serum, secondary antibody, and ABC complex from the “Elite” Vectastain ABC kit, PK-6101) (Vector Laboratories, Peterborough, U.K.). The tissue sections were then placed for 5 minutes in a 10mM phosphate buffered saline solution (pH 7.2) in which the proportion of diaminobenzidine was 0.025% and the proportion of H₂O₂ was 0.066%. The sections were given three washes in a phosphate-buffered saline solution between all incubation steps except between application of the serum and application of the primary antibody. After the development of the diaminobenzidine, the sections were washed in tap water for 10 minutes, and the procedure was repeated with a second monoclonal antibody to tau (mouse IgG1 diluted to a ratio of 1:4000) (Sigma-Aldrich Ltd.). The microwave retrieval was not repeated. The chromagen used was Vector Immuno Purple (Vector Laboratories), which produced an intense purple precipitate. The rater distinguished the brown-stained cells containing tryptophan hydroxylase easily from the purple-stained cells containing the tau protein, a method more reliable than any automated procedure. After they were washed with tap water, the sections were mounted in synthetic resin.

The 10-μm sections were visualized by using a Hitachi light microscope (Hitachi Denshi, Leeds, U.K.), and images were captured by using a JVC charge-coupled device (CCD) color camera (model TK-1070E) (JVC Professional, London). The areas of interest (the subdivisions of the dorsal raphe nucleus) were carefully identified, as described earlier, and demarcated under low power (×4 objective). All cells with positive staining for tryptophan hydroxylase and all cells with positive staining for tau in the demarcated areas were then counted under higher magnification (×25 objective). Thus, all cells with positive staining for tryptophan hydroxylase (the serotonergic cells) and all cells with positive staining for tau (cells with neuritic pathology) in each of the three sections of the dorsal raphe nucleus in each subject were counted. The mean number of serotonergic neurons counted per subject was 318. A mean count per unit area was obtained for both cell types, and the proportion of tau-positive serotonergic cells in each of the three sections was also calculated. Reliability testing of the image analysis was performed by using the data for five randomly chosen subjects, who were assessed on three separate occasions. The procedure had high intrarater reliability (coefficient of variation): 1.6% for evaluation of serotonergic cell density (cells/mm²), 1.6% for evaluation of tau-positive cell density (cells/mm²), and 2.1% for the evaluation of proportion of tau-positive cells.

All statistical analyses were performed by using the SPSS software program, version 9.0 (SPSS, Chicago). The Shapiro-Wilk test of normality and Levene’s test of homogeneity of variances were performed to determine the normality of the data and the equivalence of variances, respectively. Square root transformations were performed if necessary, and comparisons were made by using analysis of variance (ANOVA), which was followed by post hoc unpaired t tests or chi-square tests, as appropriate. The effects of possible confounders were explored by using ANOVA and Pearson’s product-moment correlation.

The three groups differed in serotonergic cell density, tau-positive cell density, and the proportion of tau-positive cells (Table 1). Post hoc testing revealed that the Alzheimer’s disease subjects had a significantly lower mean serotonergic cell density—about 45% lower—than the comparison subjects and the depressed subjects. In contrast, the Alzheimer’s disease subjects had significantly higher (about ninefold higher) mean tau-positive cell density and a significantly higher proportion of tau-positive cells, relative to the comparison subjects and the depressed subjects. Tau-positive cell density was highly negatively correlated with serotonergic cell density (r=–0.57, df=37, p<0.001). Post hoc testing revealed no significant differences in tau-positive cell density between the comparison group and the depressed group.

We carefully examined possible confounding effects on these findings. Age was correlated with serotonergic cell density (r=–0.39, df=37, p<0.02) and with tau-positive cell density (r=0.38, df=37, p<0.02), and tau-positive cell density was correlated with duration of tissue fixation (r=–0.34, df=37, p<0.04), but both age and duration of fixation were included as covariates in the analyses reported earlier. No correlations were found between postmortem interval and either serotonergic cell density or tau-positive cell density (r<0.14, df=37, p>0.38). Within the depressed group there was no correlation of either serotonergic cell density or tau-positive cell density with age at onset of depression (r<0.05, df=11, p>0.80) or any differences in serotonergic cell density or tau-positive cell density between those with an early onset of depression (before age 60 years, N=5) and those with a late onset (N=9) (F<1.70, df=11, p>0.23). We also examined the effects of a history of treatment with psychotropic medication (tricyclic antidepressants, selective serotonin reuptake inhibitors, antipsychotic drugs, and benzodiazepines) and ECT on the findings, but we found no evidence of any effect of treatment on either tau-positive cell density (F<1.79, df=1, 12, p>0.21) or serotonergic cell density (F<0.26, df=1, 12, p>0.85).

Each of the four subnuclei examined showed a similar pattern to that found for the whole dorsal raphe nucleus. These data are presented in Table 2.

The 15 Alzheimer’s disease subjects consisted of eight subjects who had comorbid depression and seven subjects with no depression (Table 3). Analysis revealed no significant differences between the two groups in sex, postmortem interval, or duration of tissue fixation. However, the groups differed significantly in age; the subjects with Alzheimer’s disease and comorbid depression were significantly younger. Because of this difference, age was used as a covariate in the analyses of data for the Alzheimer’s disease groups.

Compared to the Alzheimer’s disease subjects without depression, the Alzheimer’s disease subjects with comorbid depression had a 34% lower mean serotonergic cell density, a 1.4-fold higher mean tau-positive cell density, and a 1.6-fold higher mean proportion of tau-positive cells in the total dorsal raphe nucleus. The difference for the mean proportion of tau-positive cells just reached significance (t=2.16, df=13, p=0.05). However, in analyses with age as a covariate, this difference was not significant (Table 3). The results for each of the four subnuclei were similar to each other (data not shown) and similar to the results for the total dorsal raphe nucleus.