Changes in activity and mRNA for rat tryptophan hydroxylase and aromatic L-amino acid decarboxylase of brain serotonergic cell bodies and terminals following neonatal 5,7-dihydroxytryptamine.

In the present study, we examined time-dependent changes in activity, mRNA and immunoreactivity of the serotonin biosynthetic enzymes, tryptophan hydroxylase (TPH) and aromatic L-amino acid decarboxylase (AADC) in dorsal raphe nucleus (DRN), caudal brainstem and hypothalamus, following intracisternal injection of 5,7-dihydroxytryptamine (5,7-DHT) in neonatal rats. TPH activity in central serotonergic cell bodies and terminals was reduced to 20-30% of control levels at 1-8 weeks after neonatal, low-dose 5,7-DHT injection (24 micrograms free base). In contrast, AADC activity was either not changed or decreased to 40% of control levels, depending on the region. In situ hybridization and immunocytochemical staining indicated that 5,7-DHT caused a marked reduction in TPH and AADC message levels as well as the number of 5-HT and AADC-immunoreactive cells within the DRN as early as 1 week after 5,7-DHT. Even 15 weeks after drug administration recovery did not occur. This apparent neuronal loss was region-specific suggesting that some serotonergic neurons are more resistant to neonatal 5,7-DHT treatment than others. Taken together, these studies indicate that neonatal treatment with 5,7-DHT produces a marked and permanent (up to 15 weeks) reduction in the number of central serotonergic neurons.

Selective effects on CRF neurons and catecholamine terminals in two stress-responsive regions of adult rat brain after prenatal exposure to diazepam.

Earlier work demonstrated that prenatal exposure to diazepam (DZ) selectively affected the noradrenergic (NE) terminals in the hypothalamus, leading to decreased basal NE levels, turnover rate, and release in adult offspring as well as altered responses to stressors in these NE projections. The exposure also affected plasma hormonal responses to stressors. In the present work, we used immunocytochemistry to study the effects of prenatal DZ exposure on NE terminals and on corticotropin-releasing factor (CRF)-containing neurons in the paraventricular nucleus (PVN) of the hypothalamus. DZ exposure (2.5 or 10 mg/kg over gestational days 14-20) led to a decrease in dopamine-beta-hydroxylase (DBH)-immunoreactivity (-ir) and a decrease in CRF-ir containing cells within the PVN of adult rats. The exposure also decreased DBH-ir in the ventral portion of the bed nucleus of the stria terminalis (BNST) but did not affect CRF-ir in the oval nucleus of BNST. Therefore, this study provides anatomic evidence that targeting benzodiazepine binding sites prenatally affects two neurotransmitter systems involved in responses to stressors.

Ontogenesis of tyrosine hydroxylase-immunopositive structures in the rat hypothalamus. Fiber pathways and terminal fields.

The innervation of the hypothalamus and septal region by catecholaminergic fibers was studied in rats from the 12th fetal day until the 9th postnatal day. Catecholaminergic fibers were visualized with preembedding immunocytochemistry using antibodies to tyrosine hydroxylase. An intensification of diaminobenzidine product with silver and gold was additionally applied to increase the sensitivity and resolution power of the routine immunocytochemical technique. It has been demonstrated that, from the 13th fetal day, the hypothalamus and the septal region receive catecholaminergic fibers either belonging to the hypothalamic neurons or coming with the medial forebrain bundle from the outside of the hypothalamus. As the development of the hypothalamus proceeds, these fibers form the extensive networks within some neurosecretory centers either containing (the zona incerta, periventricular nucleus, etc.) or almost lacking (suprachiasmatic and paraventricular nuclei) the catecholaminergic neurons. In the former case, they terminate on the processes or perikarya of catecholaminergic neurons, while in the latter case their varicosities surround the immunonegative presumptive neurons in a basket-like manner. Moreover, from the 18th fetal day catecholaminergic fibers penetrate between the ependymal cells towards the 3rd ventricle and the primary capillary plexus of the hypophysial portal circulation, apparently providing the release of catecholamines to the cerebrospinal fluid and portal blood, respectively. The data obtained in this study are considered as the morphological basis for the involvement of the hypothalamic catecholamines in neuroendocrine regulations during ontogenesis.

Creatine kinase immunoreactivity: localization in nerve terminals in the hypothalamic area and superior colliculus of the mouse brain.

Immunoperoxidase studies of mouse brain using antibody to creatine kinase (CK) revealed the presence of immunoreactive neuronal varicosities in the zona incerta, lateral hypothalamic area, arcuate nucleus and superior colliculus. In the zona incerta and lateral hypothalamic area, the varicosities were distributed among the distinctive CK-immunoreactive neurons and some varicosities apparently emerged from these neurons. In the arcuate nucleus, the varicosities were concentrated in the periventricular region and occasionally clustered around unstained neurons. In serial sections, a portion of varicosities could be traced from the lateral hypothalamic area to the arcuate nucleus. In the superior colliculus, CK immunoreactivity formed a network pattern, which was mainly confined to the stratum zonale and stratum griseum superficiale.

Tyrosine hydroxylase-immunoreactive neurons in the hypothalamus of the crested newt. An electron microscopic study.

The fine structure and specialized neuronal, vascular and ventricular relations of tyrosine hydroxylase (TH)-immunopositive neurons and processes were examined in the hypothalamus of the crested newt. TH-immunoreactive neurons form a well developed system. Its possible role in the hypothalamic neuroendocrine mechanisms is discussed.

Inhibition of monoamine oxidase in 5-hydroxytryptaminergic neurones by substituted p-aminophenylalkylamines.

A series of substituted p-aminophenethylamines and some related compounds were examined with regards to the inhibition of monoamine oxidase (MAO) in vivo inside and outside 5-hydroxytryptaminergic neurones in the rat hypothalamus. This was recorded as the protection against the irreversible inhibition of MAO produced by phenelzine by determining the remaining deaminating activity in the absence and presence of citalopram using a low (0.1 microM) concentration of [14C]-5-hydroxytryptamine (5-HT) as substrate. Some of the phenethylamines were much more potent inside than outside the 5-hydroxytryptaminergic neurones. This neuronal selectivity was antagonized by pretreatment of the rats with norzimeldine, a 5-HT uptake inhibitor, which indicates that these compounds are accumulated in the 5-HT nerve terminals by the 5-HT pump. Selectivity was obtained for compounds with dimethyl, monomethyl or unsubstituted p-amino groups. An isopropyl group appears to substitute for the dimethylamino group but with considerably lower potency. Compounds with 2-substitution showed selectivity for aminergic neurones and this effect decreased with increased size of the substituent. The 2,6-dichloro derivative FLA 365 had, however, no neuronal selective action but was a potent MAO inhibitor. Substitutions in the 3- and 5-positions decreased both potency and selectivity. Prolongation of the side chain with one methylene group abolished the preference for the MAO in 5-hydroxytryptaminergic neurones although the MAO inhibitory potency remained. The selectivity disappeared by increasing the alpha-substituent to an ethyl group but remained for the alpha,alpha-dimethyl substituted derivatives. It is concluded that compounds which are (1) transported by the 5-HT pump and (2) potent reversible MAO-A inhibitors produce pronounced inhibition of MAO in 5-hydroxytryptaminergic neurones.

Inhibitory, GABAergic nerve terminals decrease at sites of focal epilepsy.

Using an immunocytochemical method for the localization of the gamma-aminobutyric acid (GABA) synthesizing enzyme, glutamic acid decarboxylase (GAD), we have observed GABAergic nerve terminals distributed throughout all layers of normal monkey sensorimotor cortex. These terminals displayed ultrastructural characteristics that suggested that they arose from aspinous and sparsely spinous stellate neurons. In monkeys (Macaca mulatta and M. fascicularis) made epileptic by cortical application of alumina gel, a highly significant numerical decrease of GAD-positive nerve terminals occurred at sites of seizure foci indicating a functional loss of GABAergic inhibitory synapses. A loss of such inhibition at seizure foci could lead to epileptic activity of cortical pyramidal neurons.

Immunohistochemical studies on the localization and distribution of monoamine neuron systems in the rat brain. I. Tyrosine hydroxylase in the mes- and diencephalon.

The localization and distribution of tyrosine hydroxylase (TH), the first enzyme in the catecholamine synthesis, in the mes- and diencephalon has been studied with the indirect immunofluorescence technique of Coons and collaborators. Principally, TH was present in neuron systems with a distribution similar to known dopamine, noradrenaline and adrenaline systems. The present data, taken together with published and some unpublished results, indicate that all parts of most central dopamine neurons, i.e. cell body, dendrites, axon and nerve terminals, appear strongly fluorescent. The adrenaline neurons also appeared strongly fluorescent, except for their axons, which only exhibited a weak fluorescence. Only cell bodies of noradrenaline neurons were strongly fluorescent, whereas the nerve terminals and axons showed a weak or moderate fluorescence intensity. The fine noradrenaline nerve terminals in some areas, such as the thalamus, were invisible or, under favourable conditions, weakly fluorescent. Therefore, in the present study we are mainly dealing with the dopamine neurons of the upper brain stem. Our results demonstrate a widespread occurrence of TH-positive neuron systems in the mes- and diencephalon. The different mesencephalic dopamine systems and their ascending projections were visualized. Numerous TH-positive cell bodies were present along the ventricle system extending from the aqueductus cerebri to the most cranial periventricular parts of the third ventricle. The caudal part of these neurons, consisting of very small cell bodies, belong to the dorsal periventricular system described by Lindvall and Björklund. Several TH-positive cell bodies were also observed in the inferior collicle of young animals. In the superficial layers of the inferior collicles TH positive nerve terminals were seen. At the hypothalamic level the A11 to A14 cell groups as well as some additonal cell bodies and extensive nerve terminal plexuses appeared strongly fluorescent. The differences in the intensity of the TH-related immunofluorescence between various brain regions and between various neuron systems may well reflect differences in enzyme levels between the various catacholamine systems rather than be due to the existence of different types of TH.