ßCaMKII in lateral habenula mediates core symptoms of depression.

The lateral habenula (LHb) has recently emerged as a key brain region in the pathophysiology of depression. However, the molecular mechanism by which LHb becomes hyperactive in depression remains unknown. Through a quantitative proteomic screen, we found that expression of the ß form of calcium/calmodulin-dependent protein kinase type II (ßCaMΚΙΙ) was significantly up-regulated in the LHb of animal models of depression and down-regulated by antidepressants. Increasing ß-, but not α-, CaMKII in the LHb strongly enhanced the synaptic efficacy and spike output of LHb neurons and was sufficient to produce profound depressive symptoms, including anhedonia and behavioral despair. Down-regulation of ßCaMKII levels, blocking its activity or its target molecule the glutamate receptor GluR1 reversed the depressive symptoms. These results identify ßCaMKII as a powerful regulator of LHb neuron function and a key molecular determinant of depression.

The medial habenula contains a specific nonstellate subtype of astrocyte expressing the ectonucleotidase NTPDase2.

ATP-mediated synaptic transmission represents the only transmitter-gated Ca(2+)-entry pathway in neurons of the rodent medial habenula. In addition to direct purinergic receptor-mediated synaptic inputs, the medial habenula contains purinergic systems that modulate synaptic transmission. Purinergic signaling is modulated or terminated by ectonucleotidase, nucleotide-hydrolyzing enzymes of the cell surface. Here we identify the major ectonucleotidase responsible for the hydrolysis of extracellular ATP in the mouse medial habenula as ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2), using immunostaining and enzyme histochemistry. Double labeling experiments reveal that the enzyme is expressed by astrocytes enwrapping the densely packed neurons and also the myelinated fiber bundles of the stria medullaris. NTPDase2 immunoreactivity is absent from the lateral habenula. The analysis of mice expressing enhanced green fluorescent protein under the promoter of glial fibrillary acidic protein revealed that the medial habenula harbors a highly polar type of astrocytes with very long laminar cellular processes, untypical for grey matter astrocytes. Its morphology strongly differs from that of the stellate astrocytes in the adjacent lateral habenula. Our results suggest that the mouse medial habenula contains a specific perineuronal nonstellate subtype of astrocyte that expresses the ectonucleotidase NTPDase2 and is in a strategic position to modulate purinergic transmission in this subnucleus.

Morphologic and cytochemical criteria for the identification and delineation of individual subnuclei within the lateral habenular complex of the rat.

The lateral habenular complex is part of the habenular nuclei, a distinct structure in the dorsal diencephalon of all vertebrates. In contrast to the bewildering diversity of behaviors, in which the lateral habenular complex is thought to be involved, there is an astonishing lack of information concerning its cellular organization, its neuronal circuits, and the neurophysiological mechanisms, which may provide the physiological and molecular basis for its diverse biological functions. This problem may be due to an unexpected heterogeneity of the lateral habenular complex. Recently, a detailed subnuclear organization has been described (Andres et al. [1999] J Comp Neurol 407:130-150), which provides the base for a subsequent physiological and behavioral analysis of this area. Available criteria, however, can be applied to semithin sections only. To facilitate further investigations, the present work aimed to elaborate novel morphologic and immunocytochemical criteria that can be applied to conventional cryostat or Vibratome sections to allow identification and delineation of subnuclei of the lateral habenular complex. Consequently, the regional, cellular, and subcellular localization of approximately 30 different neuroactive molecules was investigated. Of these candidate molecules, gamma-aminobutyric acid-B receptor protein, Kir3.2 potassium channel protein, tyrosine hydroxylase, and neurofilament heavy chain proved to be suitable markers. Our observation suggests that the habenular subnuclei express distinct immunocytochemical characteristics. These features may be used to identify and delineate the subnuclei on conventional cryostat or Vibratome sections. From our results, it is expected that the further functional analysis of the lateral habenular complex will be facilitated considerably.

Opposite metabolic changes in the habenula and ventral tegmental area of a genetic model of helpless behavior.

Congenitally helpless rats have been selectively bred to display an immediate helpless response to stress in order to model hereditary brain differences that contribute to depression vulnerability. Differences in regional brain metabolism between congenitally helpless and non-helpless rats were investigated using quantitative cytochrome oxidase histochemistry. The results indicated that congenitally helpless rats had 64-71% elevated metabolism in the habenula and a 25% elevation in the related interpeduncular nucleus. In contrast, helpless rats had 28% reduced metabolism in the ventral tegmental area (VTA) and 14-16% reductions in the basal ganglia and basolateral and central amygdala. The opposite metabolic changes in the habenula and ventral tegmental area may be especially important for determining the congenitally helpless rat's global pattern of brain activity, which resembles the metabolic activity pattern produced by dopamine antagonism.

Elevated arylalkylamine-N-acetyltransferase (AA-NAT) gene expression in medial habenular and suprachiasmatic nuclei of hibernating ground squirrels.

Hibernation, an adaptive response for energy conservation in mammals, involves a variety of physiological changes. Melatonin is linked with the regulation of core body temperature and intervenes in generating circadian cycles; its role in seasonal (circannual) rhythms of hibernation is explored here. Melatonin is primarily produced in the pineal gland. Since arylalkylamine-N-acetyltransferase (AA-NAT) is the rate-limiting enzyme for synthesizing melatonin, AA-NAT gene expression was investigated to assess the possible role of melatonin in hibernation. The findings presented here utilized combined in situ hybridization and immunohistochemistry methodologies to evaluate the AA-NAT mRNA expression in brains of both hibernating and non-hibernating ground squirrels. Brains were examined for the expression of AA-NAT mRNA using a oligonucleotide AA-NAT probe; antibody against neurofilament-70 (NF-70) was used as a neuronal marker. All hibernating animals expressed significantly (P<0.01) elevated levels of AA-NAT mRNA in both the epithalamic medial habenular nuclei (MHb) area and the hypothalamic suprachiasmatic nuclei (SCN), which is also known as the master biologic clock. These findings represent the first demonstration of the expression of mRNA encoding for AA-NAT in the extra-pineal (i.e. SCN and MHb) sites of thirteen-lined ground squirrels and indicate that the habenular nucleus may be an important supplementary location for melatonin biosynthesis. The data presented here indicate that AA-NAT gene is one of the few specific genes up-regulated during hibernation and suggest that elevation of its expression in SCN and MHb may play an essential role in the generation and maintenance of hibernation.

Leu-enkephalin immunoreactivity in the pig pineal gland.

Leu-enkephalin-positive structures in the pig pineal gland were demonstrated immunohistochemically using mouse monoclonal antibody. The pineal glands were obtained from the newborn, 21-day and 7-month old female pig. The immunopositive nerve fibers were observed in the pineal gland as well as in the epithalamic areas. The leu-enkephalin-immunoreactive nerve fibers (single or forming small bundles) were localized mainly in the proximal part of the pineal and they were scarce in other parts. The localization of the fibers points to a central source of this innervation. The study did not show any age-dependent differences in the distribution and density of leu-ekephalin-positive nerve fibers.

Nitric oxide synthase activity reveals an asymmetrical organization of the frog habenulae during development: A histochemical and cytoarchitectonic study from tadpoles to the mature Rana esculenta, with notes on the pineal complex.

In the adult frog, structural asymmetry of the left dorsal habenula in respect to the right counterpart has been repeatedly documented in previous studies. In the present investigation, histochemical expression of beta-nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity was examined in the habenulae of the developing and adult Rana esculenta. In tadpoles and during metamorphosis, selective neuropil staining was consistently found within a lateral compartment of the medial subnucleus of the left dorsal habenula. The staining was still present in the same location, but much less intense, in the mature frog, indicating that the neurochemical pattern observed during development was at least in part transient. Thus, the present data point out a peculiar neurochemical pattern of the habenular asymmetry in the frog, suggesting that nitric oxide may be involved in the developmental shaping which leads to an asymmetrical configuration of the habenulae. In addition, NADPH-diaphorase-positive cells were detected in the frontal organ (the extracranial component of the pineal complex in strict relationship with the habenulae in the frog), and labeled fibers were found in the frontal nerve, which arises from the frontal organ. This latter finding supports the postulated relationship of the habenular asymmetry with the occurrence of the frontal organ. The finding of NADPH-diaphorase histochemical reactivity confined to a distinct portion of the medial subnucleus of the left dorsal habenula prompted a reexamination of the cytoarchitecture of the developing and mature habenular complex in the frog. The bicompartmentalization detected with histochemistry in the medial subnucleus of the left dorsal habenula of the developing and adult frog was fully supported by the study of Nissl-stained epithalamic sections. These data point out that the left-right structural differences of the frog habenular complex are more complex than previously believed, and may be subserved by chemically regulated developmental processes.

Histochemical mapping of the substrate for brain-stimulation reward with glycogen phosphorylase.

Glycogen phosphorylase is the enzyme that regulates glycogenolysis and it appears that there is a relationship between central levels of glycogen and neuronal activity, which is influenced by a variety of neurotransmitters. In the present study, glycogen phosphorylase histochemistry was used to correlate changes in metabolic activity in response to rewarding lateral hypothalamic stimulation. Rats were allowed to self-stimulate for 1 h per day for ten consecutive days following which postmortem phosphorylase a activity was examined. Significant differences in optical density between the stimulated and contralateral hemispheres were found in three of the eight analyzed structures, two of which, the diagonal band of Broca and the caudate nucleus, showed a greater density of glycogen phosphorylase a on the stimulated side and the third, the habenula, had greater contralateral activity. In conclusion, our data suggest that glycogen phosphorylase activity is a viable but not weighty marker of energy alterations induced by chronic exposure to intracranial self-stimulation, and that it is generally consistent with the patterns revealed by other metabolic indices such as cytochrome oxidase and 2-deoxyglucose autoradiography.

Morphological and immunocytochemical features of the pineal organ of C3H and C57BL mice at different stages of postnatal development.

Considerable progress is currently being made in elucidating the molecular basis of the circadian (photoneuroendocrine) system by use of transgenic mice generated from the inbred strains C57BL and C3H. As in all other vertebrate species, the pineal organ is an important component of the photoneuroendocrine system in these mouse strains, but very little is known about its morphological and immunocytochemical features. We therefore investigated the pineal organ and the adjacent epithalamic region of adult, 10-, and 5-day-old C57BL and C3H mice for S-antigen, serotonin, and dopamine-ss-hydroxylase (DBH) immunoreactions. In adult animals, the pineal organ was more than 2 times bigger in C3H than in C57BL mice. In younger animals, this difference was already evident, but less pronounced. The S-antigen immunoreactivity was more intense in adult C3H than in C57BL mice. This difference developed with increasing age; it was not yet detectable in 5-day-old animals. The intensity of the serotonin immunoreaction was similar in both strains at all stages investigated. However, the serotonin immunoreaction was more pronounced in adult than in young animals. The relative DBH-immunoreactive area (used as a marker for the sympathetic innervation of the pineal organ) was much bigger in C3H than in C57BL mice; within each strain it remained relatively constant during postnatal development. Adult individuals of both strains contained S-antigen- and serotonin-immunoreactive cells in the habenular complex. Their number increased with age, but they were always more numerous in C3H. In conclusion, the study has shown considerable differences in pineal morphology between C3H and C57BL, which may be related to the well-known differen- ces in melatonin formation between these two strains.

Comparison of tyrosine hydroxylase immunoreactivity in cochleas of chinchilla and guinea pig.

OBJECTIVE: To investigate the distribution of the tyrosine hydroxylase (TH) immunoreactivity in the cochleas of chinchilla and compare it with guinea pig. METHODS: Ten healthy chinchillas and six healthy guinea pigs were used in this study. The surface preparation of cochleas were obtained by microdissection. The TH-like immunoreactivity on the surface preparation as examined by the avidin and biotinylated horseradish peroxidase macromolecular complex (ABC) technique and investigated with light microscopy. RESULTS: TH-like immunoreactivity was seen in the region from modiolus to the habenula perforata. There are two different groups of fibers in this area, one is blood vessel dependent, the other is a blood vessel independent system. No TH-like immunoreactivity was present in the inner spiral bundle (ISB) and the tunnel spiral bundle (TSB) regions in chinchilla cochleas. TH-like immunoreactivity was found in the region from modiolus to the habenula perforata and the ISB and TSB regions in the organ of corti (OC) in guinea pig cochleas. CONCLUSIONS: The presence of TH-immunoreactivity on the cochlea of chinchilla and guinea pig is different. TH-like immunoreactivity on chinchilla cochlea was involved only in the fibers corresponding to the sympathetic fibers on cochlea of guinea pig. No fibers corresponding to the dopaminergic olivocochlear lateral efferent system of guinea pig were found in ISB and TSB within the OC in chinchilla.

Immunohistochemical localization of ADP-ribosylarginine hydrolase in rodent CNS.

Polyclonal antibodies were generated against ADP-ribosylarginine hydrolase (AAH), using recombinant fusion protein of rat AAH and glutathione-S-transferase as a immunogen, and affinity-purified. Western blotting showed that the antibodies recognized in mouse brain homogenate a single protein with a molecular mass of 38 kDa, the expected size for mouse AAH. An analysis using the antibodies revealed that heavy labelings were apparent in various brain regions. In the cerebral cortex, pyramidal cells in layers III and V were the most heavily labeled. In the hippocampal formation, labeling was present on the pyramidal neurons and granule cells. The most heavily immunostained cell type was the pyramidal neuron of CA3. In the cerebellum, Purkinje cells were the most heavily labeled. Less intense staining was present over the granule cells. In the basal ganglia, neurons in the caudate nucleus and large multipolar cells in the amygdaloid complex were immunoreactive. Heavy labeling was seen in many midbrain and brainstem nuclei. Neurons in the habenula and ependymal cells were stained heavily. On Western blot analysis of rat cerebrospinal fluid (CSF), the anti-AAH antibodies recognized a protein with a molecular mass of 38 kDa. This is apparently the first evidence of a widespread but distinctive distribution of AAH in neurons of mouse brain and the presence of extracellular AAH in rat CSF.

Habrec1, a novel serine/threonine kinase TGF-beta type I-like receptor, has a specific cellular expression suggesting function in the developing organism and adult brain.

Members of the TGF-beta superfamily signal through a dual receptor system consisting of a type II receptor protein kinase that binds the ligand, after which this complex associates with a type I receptor to mediate intracellular signaling. In mammals, six type I and five type II receptors mediating responses to different TGF-beta family members have been identified to date. Using primers from conserved regions of the protein kinase domain of the serine/threonine kinase receptors in a low-stringency polymerase chain reaction-based screening procedure, and deselecting known receptors with colony hybridization, we now report cloning a novel receptor member. The novel receptor was found in a cDNA library prepared from the habenular nucleus area and was designated Habrec1. Although only a partial sequence is available, it fits the criteria for a TGF-beta type I serine/threonine kinase receptor. In situ hybridization of Habrec1 reveals mRNA expression in several distinct areas of the developing central nervous system, including cortex cerebri, cerebellum, hippocampus, striatum, and thalamic nuclei. Expression is also seen in the anterior pituitary. In the periphery, strong expression prenatally includes brown fat, the gastrointestinal tract, liver, pancreas, thymus, and nasal cavity epithelium. In the adult brain Habrec1 mRNA is prominently found in cerebellum, cortex cerebri, and striatum, but at lower levels in several additional areas. We conclude that Habrec1 is a member of the TGF-beta type I receptor family with expression patterns in the developing animal, suggesting specific functions in and outside the nervous system, and in the adult CNS, suggesting roles in both cortical and subcortical brain circuitry.

Qualitative enzyme histochemistry and microanalysis reveals changes in ultrastructural distribution of calcium and calcium-activated ATPases after microwave irradiation of the medial habenula.

The localization of calcium and calcium-activated ATPases was investigated electron microscopically in the medial habenula of mice after whole body irradiation with modulated microwaves. In non-irradiated animals calcium-containing precipitates were seen in different subcellular compartments and were often localized on the luminal side of membranes of synaptic vesicles in nerve terminals. At 1 h after 16-Hz modulated microwave irradiation, the number of synaptic vesicles containing calcium precipitates decreased, and reaction products appeared at new locations: in the synaptic clefts and on non-synaptic surfaces of the neuronal plasma membrane. This modified calcium distribution remained unchanged for 24 h following irradiation. Calcium-activated "ecto"-localized ATPase was detected as a punctuated-linear distribution of the reaction product outlining whole areas of glial and neuronal plasma membrane in the habenula of control animals. This pattern did not change on microwave irradiation. However, a quercetin-sensitive "endo"-localized Ca(2+)-ATPase activity appeared in some nerve terminals 24 h after irradiation. Thus, microwave irradiation can influence neuronal calcium homeostasis by inducing Ca2+ redistribution across the plasma membrane and by modifying Ca(2+)-ATPase activity. However, no direct correlation between these effects could be demonstrated by the present study.