Rawien Balesar

Distribution of MT1 melatonin receptor immunoreactivity in the human hypothalamus and pituitary gland: colocalization of MT1 with vasopressin, oxytocin, and corticotropin-releasing hormone.

Melatonin is implicated in numerous physiological processes, including circadian rhythms, stress, and reproduction, many of which are mediated by the hypothalamus and pituitary. The physiological actions of melatonin are mainly mediated by melatonin receptors. We here describe the distribution of the melatonin receptor MT1 in the human hypothalamus and pituitary by immunocytochemistry. MT1 immunoreactivity showed a widespread pattern in the hypothalamus. In addition to the area of the suprachiasmatic nucleus (SCN), a number of novel sites, including the paraventricular nucleus (PVN), periventricular nucleus, supraoptic nucleus (SON), sexually dimorphic nucleus, the diagonal band of Broca, the nucleus basalis of Meynert, infundibular nucleus, ventromedial and dorsomedial nucleus, tuberomamillary nucleus, mamillary body, and paraventricular thalamic nucleus were observed to have neuronal MT1 receptor expression. No staining was observed in the nucleus tuberalis lateralis and bed nucleus of the stria terminalis. The MT1 receptor was colocalized with some vasopressin (AVP) neurons in the SCN, colocalized with some parvocellular and magnocellular AVP and oxytocine (OXT) neurons in the PVN and SON, and colocalized with some parvocellular corticotropin‐releasing hormone (CRH) neurons in the PVN. In the pituitary, strong MT1 expression was observed in the pars tuberalis, while a weak staining was found in the posterior and anterior pituitary. These findings provide a neurobiological basis for the participation of melatonin in the regulation of various hypothalamic and pituitary functions. The colocalization of MT1 and CRH suggests that melatonin might directly modulate the hypothalamus–pituitary–adrenal axis in the PVN, which may have implications for stress conditions such as depression. J. Comp. Neurol. 499:897–910, 2006.

Analysis of Gene Expression in Parkinson's Disease : Possible Involvement of Neurotrophic Support and Axon Guidance in Dopaminergic Cell Death

Parkinsons disease (PD) is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra. We have studied alterations in gene expression in the substantia nigra, the caudate nucleus and putamen of four PD patients and four matched controls using custom designed Agilent microarrays. To gain insight into changes in gene expression during early stages of dopaminergic neurodegeneration, we selectively investigated the relatively spared parts of the PD substantia nigra, and correlated gene expression changes with alterations in neuronal density. We identified changes in the expression of 287 transcripts in the substantia nigra, 16 transcripts in the caudate nucleus and four transcripts in the putamen. For selected transcripts, transcriptional alterations were confirmed with qPCR on a larger set of seven PD cases and seven matched controls. We detected concerted changes in functionally connected groups of genes. In the PD substantia nigra, we observed strong evidence for a reduction in neurotrophic support and alterations in axon guidance cues. As the changes occur in relatively spared parts of the PD substantia nigra, they suggest novel disease mechanisms involving neurotrophic support and axon guidance in early stages of cellular stress events, ultimately leading to dopaminergic cell death in PD.

Estrogen receptor‐α distribution in the human hypothalamus in relation to sex and endocrine status

The present study reports the first systematic rostrocaudal distribution of estrogen receptor‐α immunoreactivity (ERα‐ir) in the human hypothalamus and its adjacent areas in young adults. Postmortem material taken from 10 subjects (five male and five female), between 20 and 39 years of age, was investigated. In addition, three age‐matched subjects with abnormal levels of estrogens were studied: a castrated, estrogen‐treated 50‐year‐old male‐to‐female transsexual (T1), a 31‐year‐old man with an estrogen‐producing tumor (S2), and an ovariectomized 46‐year‐old woman (S8). A strong sex difference, with more nuclear ERα‐ir in women, was observed rostrally in the diagonal band of Broca and caudally in the medial mamillary nucleus. Less robust sex differences were observed in other brain areas, with more intense nuclear ERα‐ir in men, e.g., in the sexually dimorphic nucleus of the medial preoptic area, paraventricular nucleus, and lateral hypothalamic area, whereas women had more nuclear ERα‐ir in the suprachiasmatic nucleus and ventromedial nucleus. No nuclear sex differences in ERα were found, e.g., in the central part of the bed nucleus of the stria terminalis. In addition to nuclear staining, ERα‐ir appeared to be sex‐dependently present in the cytoplasm of neurons and was observed in astrocytes, plexus choroideus, and other non‐neuronal cells. ERα‐ir in T1, S2, and S8 suggested that most of the observed sex differences in ERα‐ir are “activational” (e.g., ventromedial nucleus/medial mamillary nucleus) rather than “organizational.” Species similarities and differences in ERα‐ir distribution and possible functional implications are discussed. J. Comp. Neurol. 454:115–139, 2002.

Estrogen‐receptor‐β distribution in the human hypothalamus: Similarities and differences with ERα distribution

This study reports the first systematic rostrocaudal distribution of estrogen receptor beta immunoreactivity (ERβ‐ir) in the human hypothalamus and adjacent areas in five males and five females between 20–39 years of age and compares its distribution to previously reported ERα in the same patients. ERβ‐ir was generally observed more frequently in the cytoplasm than in the nucleus and appeared to be stronger in women. Basket‐like fiber stainings, suggestive for ERβ‐ir in synaptic terminals, were additionally observed in various areas. Men showed more robust nuclear ERβ‐ir than women in the medial part of the bed nucleus of the stria terminalis, paraventricular and paratenial nucleus of the thalamus, while less intense, but more nuclear, ERβ‐ir appeared to be present in, e.g., the BSTc, sexually dimorphic nucleus of the medial preoptic area, diagonal band of Broca and ventromedial nucleus. Women revealed more nuclear ERβ‐ir than men of a low to intermediate level, e.g., in the suprachiasmatic, supraoptic, paraventricular, infundibular, and medial mamillary nucleus. These data indicate potential sex differences in ERβ expression. ERβ‐ir expression patterns in subjects with abnormal hormone levels suggests that there may be sex differences in ERβ‐ir that are “activational” rather than “organizational” in nature. Similarities, differences, potential functional, and clinical implications of the observed ERα and ERβ distributions are discussed in relation to reproduction, autonomic‐function, mood, cognition, and neuroprotection in health and disease. J. Comp. Neurol. 466:251–277, 2003.

A direct androgenic involvement in the expression of human corticotropin-releasing hormone

We investigated the possibility of a direct action of androgens on the expression of the human corticotropin-releasing hormone (CRH), which plays a central role in the hypothalamic–pituitary–adrenal (HPA)-axis. Colocalization of CRH and nuclear/cytoplasmic androgen receptor (AR) was found in neurons of the paraventricular nucleus (PVN) in the human hypothalamus. A potential androgen-responsive element (ARE) in the human CRH promoter was subsequently analyzed with bandshifts and cotransfections in neuroblastoma cells. In the presence of testosterone, recombinant human AR bound specifically to the CRH-ARE. Expression of AR in combination with testosterone repressed CRH promoter activity through the ARE. We conclude that androgens may directly affect CRH neurons in the human PVN via AR binding to the CRH-ARE, which may have consequences for sex-specific pathogenesis of mood disorders.

Alterations in the histaminergic system in the substantia nigra and striatum of Parkinson's patients: a postmortem study

Earlier studies showed neuronal histamine production in the hypothalamic tuberomamillary nucleus to be unchanged in Parkinsons disease (PD), whereas the histamine levels and innervation in the substantia nigra (SN) increased. In the present study we used quantitative polymerase chain reaction (qPCR) to assess the changes in the histaminergic system in the SN, caudate nucleus (CN), and putamen (PU) in 7 PD patients and 7 controls. The messenger RNA (mRNA) expression of the histamine receptor-3 (H(3)R), which was localized immunocytochemically in the large pigmented neurons, was significantly decreased in the SN in PD, while histamine receptor-4 (H(4)R)-mRNA expression showed a significant increase in caudate nucleus and PU. In addition, significantly increased mRNA levels of histamine methyltransferase (HMT), a key enzyme involved in histamine metabolism, were found in the SN and in the PU in PD. Moreover, in the SN, the histamine methyltransferase-mRNA showed a strong negative correlation with PD disease duration. Our observations imply the presence of local changes in the histaminergic system that may contribute to PD pathology, and may thus provide a rationale for possible novel therapeutic strategies.

Decreased NOS1 Expression in the Anterior Cingulate Cortex in Depression

Decreased function of the anterior cingulate cortex (ACC) is crucially involved in the pathogenesis of depression. A key role of nitric oxide (NO) has also been proposed. We aimed to determine the NO content in the cerebrospinal fluid (CSF) and the expression of NO synthase (NOS) isoforms, that is, NOS1, NOS2, and NOS3 in the ACC in depression. In depressive patients, CSF-NOx levels (the levels of the NO metabolites nitrite and nitrate) were significantly decreased (P = 0.007), indicating a more general decrease of NO production in this disorder. This agreed with a trend toward lower NOS1-mRNA levels (P = 0.083) and a significant decrease of NOS1-immunoreactivity (ir) (P = 0.043) in ACC. In controls, there was a significant positive correlation between ACC-NOS1-ir cell densities and their CSF-NOx levels. Furthermore, both localization of NOS1 in pyramidal neurons that are known to be glutamatergic and co-localization between NOS1 and GABAergic neurons were observed in human ACC. The diminished ACC-NOS1 expression and decreased CSF-NOx levels may be involved in the alterations of ACC activity in depression, possibly by affecting glutamatergic and GABAergic neurotransmission.

Neural stem cells improve neuronal survival in cultured postmortem brain tissue from aged and Alzheimer patients

Neurodegenerative diseases are progressive and incurable and are becoming ever more prevalent. To study whether neural stem cell can reactivate or rescue functions of impaired neurons in the human aging and neurodegenerating brain, we co‐cultured postmortem slices from Alzheimer patients and control participants with rat embryonic day 14 (E14) neural stem cells. Viability staining based on the exclusion of ethidium bromide by intact plasma membranes showed that there were strikingly more viable cells and fewer dead cells in slices co‐cultured with neural stem cells than in untreated slices. The presence of Alzheimer pathology in the brain slices did not influence this effect, although the slices from Alzheimer patients, in general, contained fewer viable cells. Co‐culturing with rat E14 fibroblasts did not improve the viability of neurons in the human brain slices. Since the human slices and neural stem cells were separated by a membrane during co‐culturing our data show for the first time that neural stem cells release diffusible factors that may improve the survival of aged and degenerating neurons in human brains.

Neuronal histamine production remains unaltered in Parkinson's disease despite the accumulation of Lewy bodies and Lewy neurites in the tuberomamillary nucleus

Neuronal histamine production in the hypothalamic tuberomamillary nucleus (TMN) was hypothesized to change significantly in Parkinsons disease (PD) in relation to the accumulation of Lewy bodies/Lewy neurites (LBs/LNs). We measured the messenger ribonucleic acid (mRNA) levels of histidine decarboxylase (HDC), the key enzyme of histamine production, and the amount of LBs/LNs in the TMN by quantitative in situ hybridization and immunocytochemistry in postmortem human brain material of clinical PD (CPD), preclinical PD, and control subjects. No significant difference of histidine decarboxylase mRNA levels was observed among different clinical or Braak-PD stages, in spite of the strong accumulation of LBs/LNs in the TMN of clinical PD patients. We conclude that neuronal histamine production remains largely unaltered in PD despite the abundant LB/LN accumulation in the TMN.

A quantitative in situ hybridization protocol for formalin-fixed paraffin-embedded archival post-mortem human brain tissue

The use of radioactive in situ hybridization (ISH) to quantitatively determine low-to-moderate abundant mRNA expression in formalin-fixed, paraffin-embedded archival post-mortem human brain tissue is often limited by non-specific-deposits, visible as speckles. In the present study, optimal hybridization conditions were achieved for quantifying the mRNA expression of histidine decarboxylase (HDC) by a number of alterations in a routine protocol, which included (1) during purification of the oligo-probes, glycogen was omitted as a carrier for precipitation, (2) after precipitation, the labeled probe contained within the pellet was first dissolved in water instead of in hybridization buffer (HBF), (3) during hybridization, the dithiothreitol (DTT) concentration was increased from 200 to 800 mM in HBF, and (4) stringencies during hybridization and post-hybridization washes were increased by increasing the temperature. The effect of the adjustment was quantified on adjacent sections from 18 subjects (9 with Parkinsons disease and 9 controls), by comparing the data from the standard and new protocol. The results showed that the improved protocol brought about significantly clearer background with higher signal-to-noise ratios (p=0.001). We propose that this protocol is also applicable for detection of other lower-abundant genes in human brain tissue and probably in other tissues as well. In the present study, this is not only illustrated for HDC ISH, but also for corticotrophin-releasing hormone mRNA expression in the hypothalamic paraventricular nucleus.