Nuclear organization of the substantia nigra, ventral tegmental area and retrorubral field of the common marmoset (Callithrix jacchus): A cytoarchitectonic and TH-immunohistochemistry study.

It is widely known that the catecholamine group is formed by dopamine, noradrenaline and adrenaline. Its synthesis is regulated by the enzyme called tyrosine hydroxylase. 3-hydroxytyramine/dopamine (DA) is a precursor of noradrenaline and adrenaline synthesis and acts as a neurotransmitter in the central nervous system. The three main nuclei, being the retrorubral field (A8 group), the substantia nigra pars compacta (A9 group) and the ventral tegmental area (A10 group), are arranged in the die-mesencephalic portion and are involved in three complex circuitries - the mesostriatal, mesolimbic and mesocortical pathways. These pathways are involved in behavioral manifestations, motricity, learning, reward and also in pathological conditions such as Parkinson's disease and schizophrenia. The aim of this study was to perform a morphological analysis of the A8, A9 and A10 groups in the common marmoset (Callithrix jacchus - a neotropical primate), whose morphological and functional characteristics support its suitability for use in biomedical research. Coronal sections of the marmoset brain were submitted to Nissl staining and TH-immunohistochemistry. The morphology of the neurons made it possible to subdivide the A10 group into seven distinct regions: interfascicular nucleus, raphe rostral linear nucleus and raphe caudal linear nucleus in the middle line; paranigral and parainterfascicular nucleus in the middle zone; the rostral portion of the ventral tegmental area nucleus and parabrachial pigmented nucleus located in the dorsolateral portion of the mesencephalic tegmentum. The A9 group was divided into four regions: substantia nigra compacta dorsal and ventral tiers; substantia nigra compacta lateral and medial clusters. No subdivisions were made for the A8 group. These results reveal that A8, A9 and A10 are phylogenetically stable across species. As such, further studies concerning such divisions are necessary in order to evaluate the occurrence of subdivisions that express DA in other primate species, with the aim of characterizing its functional relevance.

A forebrain-retrorubral pathway involved in male sex behavior is GABAergic and activated with mating in gerbils.

The ventral bed nuclei of the stria terminalis (BST) and medial preoptic nucleus (MPN) of gerbils contain cells that regulate male sex behavior via a largely uncrossed pathway to the retrorubral field (RRF). Our goal was to learn more about cells at the pathway source and target. To determine if the pathway uses GABA as its transmitter, we used immunocytochemistry (ICC) to study glutamic acid decarboxlyase(67) (GAD(67)) colocalization with fluoro-gold (FG) in the ventral BST and MPN after applying FG to the RRF. To determine if the pathway is activated with mating, we studied FG-Fos colocalization in the ventral BST of recently mated males. The ventral BST expresses Fos with mating and is the major pathway source. To determine to what extent other GABAergic cells in the ventral BST are activated with mating, we studied Fos colocalization with GAD(67) mRNA visualized by in situ hybridization (ISH). We also looked for GAD(67) mRNA in RRF cells. Almost all ventral BST and MPNm cells projecting to the RRF (95-97%) and most ventral BST cells activated with mating (89%), were GABAergic. GABAergic cells were also seen in the RRF. RRF-projecting cells represented 37% of ventral BST cells activated with mating. Their activation may reflect arousal and anticipation of sexual reward. Among ventral BST cells that project to the RRF, 14% were activated with mating, consistent with how much of this pathway is needed for mating. The activated GABAergic cells that do not project to the RRF may release GABA locally and inhibit ejaculation.

First visualization of cholinergic cells and fibers by immunohistochemistry for choline acetyltransferase of the common type in the optic lobe and peduncle complex of Octopus vulgaris.

This study provides the first immunohistochemical evidence visualizing cholinergic octopus neurons containing choline acetyltransferase (ChAT), the synthetic enzyme of acetylcholine. Because the antiserum applied here was raised against a recombinant protein encoded by exons 7 and 8 of the rat gene for ChAT, and initially used for studies in mammals, to validate antibody specificity for the octopus counterpart enzyme we therefore used three methods. Immunoprecipitation using Pansorbin indicated that immunoreactive octopus brain molecules were capable of synthesizing acetylcholine. Western blot analysis after denatured gel electrophoresis of octopus brain extracts revealed a single band at approximately 81 kDa. A gel slice containing the 81-kDa protein after native (nondenatured) gel electrophoresis exhibited high ChAT activity. All findings obtained with these three methods clearly indicated that the antiserum effectively recognizes octopus ChAT. The immunohistochemical use of the antiserum in the retina, optic lobe, and its neighboring peduncle complex detected enzyme-containing neuronal cell bodies in only two regions, the cell islands of the optic lobe medulla and the cortical layer of the posterior olfactory lobule. Immunoreactive fibers and probable nerve terminals were also found in the plexiform layer of the deep retina, within the stroma of the optic gland, and the neuropils of the optic lobe, peduncle lobe, and olfactory lobe. These results provide information on the morphology and distribution patterns of cholinergic neurons in the octopus visual system, a useful invertebrate model for learning and memory where the cholinergic system, as in higher vertebrates including mammals, plays an important role.

Long-term upregulation of protein kinase A and adenylate cyclase levels in human smokers.

Repeated injections of cocaine and morphine in laboratory rats cause a variety of molecular neuroadaptations in the cAMP signaling pathway in nucleus accumbens and ventral tegmental area. Here we report similar neuroadaptations in postmortem tissue from the brains of human smokers and former smokers. Activity levels of two major components of cAMP signaling, cAMP-dependent protein kinase A (PKA) and adenylate cyclase, were abnormally elevated in nucleus accumbens of smokers and in ventral midbrain dopaminergic region of both smokers and former smokers. Protein levels of the catalytic subunit of PKA were correspondingly higher in the ventral midbrain dopaminergic region of both smokers and former smokers. Protein levels of other candidate neuroadaptations, including glutamate receptor subunits, tyrosine hydroxylase, and other protein kinases, were within normal range. These findings extend our understanding of addiction-related neuroadaptations of cAMP signaling to tobacco smoking in human subjects and suggest that smoking-induced brain neuroadaptations can persist for significant periods in former smokers.

Mouse tryptophan hydroxylase isoform 2 and the role of proline 447 in enzyme function.

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of the neurotransmitter serotonin (5-HT). Once thought to be a single gene product, TPH is now known to exist in two isoforms. Isoform 1 (TPH1) is found in the pineal gland and gut, and isoform 2 (TPH2) is selectively expressed in brain. A single-nucleotide polymorphism in TPH2 results in a proline-to-arginine mutation at residue 447 and substantially lowers catalytic activity. In view of the importance of TPH in determining brain 5-HT function, we cloned TPH2 and produced the P447R mutant to assess the importance of this proline in enzyme function. Catalytically active TPH2 and the P447R mutant were expressed at the predicted subunit molecular mass of 56 kDa. The P447R mutant expressed less than 50% of the activity of TPH2. Mutation of this conserved proline in TPH1 (P403R) also resulted in an enzyme with significantly lower activity than the wild-type enzyme. The P447R mutant had a V(max) 50% lower than that of TPH2. The P447R mutation did not alter the oligomeric assembly of the protein, nor change its responsiveness to cysteine modification. The P447R mutation did not alter enzyme substrate specificity or stability, but conferred slightly enhanced sensitivity to inhibition by dopamine and diminished sensitivity to iron in catalysis. The conserved proline in TPH (residue 447 in TPH2 and 403 in TPH1) plays an important role in enzyme function by regulating V(max) of the catalytic reaction.

Increased levels of tyrosine hydroxylase and glutamic acid decarboxylase in locus coeruleus neurons after rapid eye movement sleep deprivation in rats.

Norepinephrine, acetylcholine and GABA levels alter during rapid eye movement (REM) sleep and its deprivation. Increased synthesis of those neurotransmitters is necessary for their sustained release. Hence, in this study, the concentrations of tyrosine hydroxylase (TH), choline acetyl transferase (ChAT) and glutamic acid decarboxylase (GAD), the enzymes responsible for their synthesis, were immunohistochemically estimated within the neurons in locus coeruleus, laterodorsal tegmentum and pedunculopontine tegmentum and medial preoptic area in REM sleep deprived and control rats. It was observed that as compared to controls, deprivation increased TH and GAD significantly in the locus coeruleus only, while in other areas, they remained unchanged. The findings help explaining the mechanism of increase in neurotransmitter levels in the brain after REM sleep deprivation and their significance has been discussed.

GABAergic neurons of the laterodorsal and pedunculopontine tegmental nuclei of the cat express c-fos during carbachol-induced active sleep.

The laterodorsal and pedunculopontine tegmental nuclei (LDT-PPT) are involved in the generation of active sleep (AS; also called REM or rapid eye movement sleep). Although the LDT-PPT are composed principally of cholinergic neurons that participate in the control of sleep and waking states, the function of the large number of GABAergic neurons that are also located in the LDT-PPT is unknown. Consequently, we sought to determine if these neurons are activated (as indicated by their c-fos expression) during active sleep induced by the microinjection of carbachol into the rostro-dorsal pons (AS-carbachol). Accordingly, immunocytochemical double-labeling techniques were used to identify GABA and Fos protein, as well as choline acetyltransferase (ChAT), in histological sections of the LDT-PPT. Compared to control awake cats, there was a larger number of GABAergic neurons that expressed c-fos during AS-carbachol (31.5+/-6.1 vs. 112+/-15.2, P<0.005). This increase in the number of GABA+Fos+ neurons occurred on the ipsilateral side relative to the injection site; there was a small decrease in GABA+Fos+ cells in the contralateral LDT-PPT. However, the LDT-PPT neurons that exhibited the largest increase in c-fos expression during AS-carbachol were neither GABA+ nor ChAT+ (47+/-22.5 vs. 228.7+/-14.0, P<0.0005). The number of cholinergic neurons that expressed c-fos during AS-carbachol was not significantly different compared to wakefulness. These data demonstrate that, during AS-carbachol, GABAergic as well as an unidentified population of neurons are activated in the LDT-PPT. We propose that these non-cholinergic LDT-PPT neurons may participate in the regulation of active sleep.

The effect of electrolytic thalamic lesions on the NADPH-diaphorase activity of neurons of the laterodorsal tegmental and pedunculopontine nuclei in rats.

Cholinergic neurons of the mesopontine complex have extensive ascending projections to the forebrain: the laterodorsal tegmental nucleus extensively innervates the anterior thalamus, the anteroventral nucleus in particular, whereas the pedunculopontine nucleus has widespread projections to both the thalamus and extrapyramidal structures. Most of their neurons express nitric oxide synthase (NOS) activity. Following electrolytic lesions of the anteroventral thalamic nucleus, nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) activity in neurons of the laterodorsal tegmental nucleus changed drastically. The intensity of NADPH-diaphorase staining increased in laterodorsal tegmental neurons ipsilateral to the lesion side, but decreased contralaterally. The intensity of the NADPH-diaphorase staining of neurons of the pedunculopontine nucleus, however, remained unchanged bilaterally. After partial lesions of the anteroventral thalamic nucleus a similar effect was noted. In contrast, large electrolytic lesions involving other thalamic nuclei or extrapyramidal structures did not change the number of NADPH-diaphorase neurons or their intensity of staining in the laterodorsal tegmental nuclei. These data show that electrolytic lesions of target areas can lead to an upregulation of NOS expression in the parent cell bodies, provided that there is no wide collateralization as found for the pedunculopontine nucleus.

Ultrastructural localization of neuronal nitric oxide synthase in the laterodorsal tegmental nucleus of wild-type and knockout mice.

The cellular and subcellular distribution of neuronal nitric oxide synthase and its related reduced beta-nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity was compared in wild-type and homozygous knockout mice, in which the gene for neuronal nitric oxide synthase has been disrupted, resulting in a lack of the predominant splice isoform alpha. In the laterodorsal tegmental nucleus, used as a model structure, the cholinergic principal neurons also exhibited an intensive neuronal nitric oxide synthase immunoreactivity. Using the tetrazolium salt 2-(2-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl)-tetrazo++ +-lium chloride (BSPT), these neurons were filled with NADPH-diaphorase reaction product, whereas the equivalent neurons of knockout mice showed, if at all, only traces of neuronal nitric oxide synthase immunoreactivity in parallel to a diminished NADPH-diaphorase labelling. Subcellularly, the neuronal nitric oxide synthase-related diaminobenzidine product was, apparently owing to diffusion artifact, more or less evenly distributed in the cytosol of the neuronal perikarya and dendrites of wild-type mice. In contrast, the BSPT reaction product formazan was closely and discretely attached to endocellular membranes. In the intensely NADPH-diaphorase stained neurons of wild-type mice, 85% of the mitochondria were, at least partly, labelled for BSPT-formazan, whilst in the equivalent neurons of mutant mice, only 13% of mitochondria were NADPH-diaphorase positive. Related to the NADPH-diaphorase activity in the principal neurons of wild-type mice, only 10% of membranes of the endoplasmic reticulum, 27% of mitochondrial membranes and 26% of the nuclear envelope exhibited NADPH-diaphorase activity in the mutant mice. Our findings with the BSPT histochemistry suggest that residues of NADPH-diaphorase positivity in mutant mice are attributed to the alternative splice isoforms beta and/or gamma of neuronal nitric oxide synthase. The splice isoform a is located predominantly at the membranes of the endoplasmic reticulum.

Chronic, low-level exposure to the cholinesterase inhibitor DFP. I. Time course of neurochemical changes in the rat pontomesencephalic tegmentum.

Rats were repeatedly administered with a low dose of diisopropylfluorosphosphate (DFP; 0.2 mg/kg/day, SC, for 9 or 21 days), an irreversible cholinesterase (ChE) inhibitor. Control rats received a daily injection of oil vehicle. Neurochemical changes occurring in the pontomesencephalic tegmentum (PMT), a brain stem region critically involved in behavioral state control, were evaluated at various times of treatment and after DFP withdrawal. First, enzyme assay revealed a profile of ChE inhibition in the whole PMT which looked like that observed in the striatum; both the inhibition and recovery proceeded more slowly than they did in the plasma. Second, quantitative histochemistry indicated that ChE activity in the mesopontine cholinergic nuclei and the pontine reticular formation progressively decreased across the first days of DFP exposure, to reach an asymptotic level of inhibition after 6 days (74-82% inhibition). The inhibition was less pronounced in the locus coeruleus (49%). Third, [3H]QNB autoradiography showed that muscarinic receptor density was unchanged in any of the PMT areas selected. These results are discussed regarding the question of regional variation in susceptibility to anti-ChE agents. To what extent behavioral state alterations occur concomitantly with ChE activity changes is assessed in the companion article.

Cholinergic neurons with monoamine oxidase type B (MAOB)-activity in the laterodorsal tegmental nucleus of the mouse.

No neurons in the laterodorsal tegmental nucleus (LDTg) show monoamine oxidase (MAO) activity in the rat or monkey. However, in our recent study, many LDTg neurons with MAO type B (MAOB)-activity were found in MAOA-deficient mice that were derived from C3H mouse line. In the present study, LDTg neurons with MAOB-activity were found not only in normal C3H mouse but also in BALB/C and C57BL/6 mouse lines: MAO histochemistry revealed LDTg neurons with MAO-activity even after pharmacological suppression of MAOA-activity with clorgyline, a specific MAOA inhibitor, but not after pharmacological suppression of MAOB-activity with deprenyl, a specific MAOB inhibitor. LDTg neurons with MAOB-activity also showed NADPH-diaphorase-activity, a marker of cholinergic neurons.

Regulation of phospholipase Cgamma in the mesolimbic dopamine system by chronic morphine administration.

Neurotrophic signaling pathways have been implicated in the maintenance of the mesolimbic dopamine system, as well as in changes in this system induced by chronic morphine exposure. We found that many of these signaling pathway proteins are expressed at appreciable levels within the ventral tegmental area (VTA) and related regions, although with substantial regional variation. Moreover, phospholipase Cgamma1 (PLCgamma1) was significantly and specifically up-regulated within the VTA by 30% following chronic exposure to morphine. PLCgamma1 mRNA expression is enriched in dopaminergic neurons within the VTA; however, the up-regulation of PLCgamma1 in this region was not seen at the mRNA level. In contrast to PLCgamma1, insulin receptor substrate (IRS)-2, a protein involved in phosphatidylinositol 3-kinase signaling, and another putative IRS-like protein were significantly down-regulated within the VTA by 49 and 45%, respectively. Levels of several proteins within the Ras-ERK pathway were not altered. Regulation of neurotrophic factor signaling proteins may play a role in morphine-induced plasticity within the mesolimbic dopamine system.

Neurotrophic factors increase tyrosine hydroxylase immunoreactive cell density of fetal mesencephalic cells.

OBJECTIVE: To hibernate fetal ventral mesencephalic tegmental (VMT) cells from Spraque-Dawley rats E-15 for 5 days at 4 degrees C in either hibernation media (HM) or in conditioned hibernation media (CHM) supplemented with trophic factors such as epidermal growth factor (EGF 200 ng/ml), basic fibroblast growth factor (bFGF 100 ng/ml), recombinant human brain derived neurotrophic factor (rhBDNF 20 micrograms/ml), recombinant human glial cell-derived neurotrophic factor (rhGDNF 20 micrograms/ml), fetal calf serum (FCS 9%), or human placental cord serum (HPCS 10%). METHODS: The percent of cell viability and the density of tyrosine hydroxylase immunoreactive (THir) cells in fetal striatal-VMT co-culture were determined. RESULTS: The viability of fetal striatal cells (0.8 +/- 0.104) was slightly higher than that of fetal VMT cells (0.7 +/- 0.072) at 0-time point (F17,1 = 4.677; P = 0.045). After 5 days of hibernation, the viability of fetal VMT cells reduced by 30% (F7,1 = 88.493; P < 0.001) in HM. However, THir-cell density reduced by more than 90% as compared to the freshly harvested VMT cells (F7,1 = 179.944; P < 0.001). CHM with HPCS, bFGF, EGF, BDNF, and GDNF showed higher THir-cell density than that of HM or CHM supplemented with FCS (P < 0.001). CONCLUSION: Supplementation of appropriate trophic factors for hibernated fetal VMT cells promotes cell viability and the subsequent expression of THir-cell density.

Induction of microglial reaction and expression of nitric oxide synthase I in the nucleus dorsalis and red nucleus following lower thoracic spinal cord hemisection.

In the present study, immunohistochemical stainings for OX-6, OX-42, nitric oxide synthase I and II as well as nitrotyrosine were used to investigate possible correlation among microglial reactivity, nitric oxide synthase upregulation, peroxynitrite involvement and neuronal death in the nucleus dorsalis and red nucleus following lower thoracic spinal cord hemisection. Significant neuronal loss was found in the ipsilateral nucleus dorsalis and contralateral red nucleus after cord hemisection. A distinctive microglial reaction for OX-42 could be observed from one to four weeks post axotomy in the ipsilateral nucleus dorsalis; by contrast, it was observed on both sides of the red nucleus from one to three weeks following cord hemisection. The activated microglial cells showed some degree of hypertrophy. From the microglial immunoreactivity as well as their appearance, it was speculated that microglial activation might be beneficial or protective to the axotomized neurons. In normal and sham-operated rats, neurons of the nucleus dorsalis were not nitric oxide synthase I reactive. Three weeks after cord hemisection, neurons in the ipsilateral nucleus dorsalis below the lesion showed strong immunoreactivity. Neurons in the red nucleus that normally displayed weak nitric oxide synthase I immunoreactivity showed an increase on both sides of the nucleus. These results suggested that nitric oxide synthase I expression in the nucleus dorsalis following axotomy was synthesized de novo and might act as a neurotoxic agent. However, the bilateral increase in expression of nitric oxide synthase I in the red nucleus after lower thoracic cord hemisection was due to up-regulation of the constitutive enzyme and might have some neuroprotective function. Our results also suggested that peroxynitrite played no or little role in the neurodegeneration in the nucleus dorsalis and red nucleus following axotomy.

Mesencephalic THmRNA-reduced expression by blocking axonal transport with colchicine.

Colchicine, an axonal transport blocking agent, was unilaterally injected in the medial forebrain bundle of rats. As early as 18 h after the injection a rapid decrease in TH-mRNA level was observed in the substantia nigra and the ventral tegmental area (SN/VTA) on the injected side. In contrast, TH protein levels remained stable for 48 h, and decreased later in both cells bodies and terminals (caudate/putamen). The number of TH-immunopositive cells in SN/VTA increased after colchicine equally in both sides, excluding a neurotoxic effect. These results suggest that TH gene expression is controlled by a retrogradely transported activating factor rather than by feedback inhibition by the end product, i.e. TH protein.

Haloperidol induces persistent down-regulation of tyrosine hydroxylase immunoreactivity in substantia nigra but not ventral tegmental area in the rat.

The dopamine antagonist haloperidol can cause tardive side-effects that may persist after the drug is withdrawn. We studied the time course of changes in dopaminergic neurons of the substantia nigra and ventral tegmental area following withdrawal of haloperidol. Rats received daily intraperitoneal injections of saline or haloperidol for eight weeks and were killed at two, four or 12 weeks after the final injection. Sections of substantia nigra and ventral tegmental area were processed for tyrosine hydroxylase immunohistochemistry. Quantitative morphometric analysis was carried out blinded in order to determine the number, cell body size and topography of tyrosine hydroxylase-positive cells, and the immunoreactive area of the substantia nigra and ventral tegmental area. In haloperidol-treated rats, tyrosine hydroxylase-positive cell counts were normal in ventral tegmental area but were decreased in substantia nigra by 34% at two weeks withdrawal and by 52% at four weeks withdrawal; cell counts were almost fully recovered by 12 weeks withdrawal. Cross-sectional area of tyrosine hydroxylase immunoreactivity within the substantia nigra demonstrated a similar pattern of reduction, with full recovery by 12 weeks withdrawal. Mean cell size, by contrast, was essentially unchanged at two and four weeks withdrawal, but was significantly decreased in sub-regions of substantia nigra at 12 weeks withdrawal. These results indicate that haloperidol can produce selective changes in midbrain dopamine neurons that persist long after discontinuation of the drug. This decrease in tyrosine hydroxylase-immunoreactive cell counts may play a role in the neurobiology of the persistent tardive syndromes associated with the use of neuroleptics.

L-deprenyl induces aromatic L-amino acid decarboxylase (AADC) mRNA in the rat substantia nigra and ventral tegmentum. An in situ hybridization study.

L-Deprenyl is a complex drug, and number of mechanisms have been proposed to explain its effects. These include blockade of dopamine metabolism, amplification of dopamine responses, induction of superoxide dismutase or delaying apoptosis. Using in situ hybridization techniques, we have shown that L-deprenyl (5-10 mg/kg intraperitoneally, killed after 24 h) increases aromatic L-amino acid decarboxylase (AADC) mRNA levels in rat substantia nigraventral tegmental area. In human brain tissue, AADC is present at low levels, suggesting a possible rate-limiting role in monoamine synthesis. This is particularly important in parkinsonian patients, since the therapeutic efficacy of L-DOPA is attributed to its enzymatic decarboxylation to dopamine. The present findings support that one of the effects of L-deprenyl may be to facilitate the decarboxylation of L-DOPA by increasing the availability of AADC.

Ionic mechanisms involved in the spontaneous firing of tegmental pedunculopontine nucleus neurons of the rat.

We have previously defined three types of tegmental pedunculopontine nuclei neurons based on their electrophysiological characteristics: Type I neurons characterized by low-threshold Ca2+ spikes, Type II neurons which displayed a transient outward current (A-current), and Type III neurons having neither low-threshold spikes nor A-current [Kang Y. and Kitai S. T. (1990) Brain Res. 535, 79-95]. In this report, ionic mechanisms underlying repetitive firing of Type I (n=15) and Type II (n=69) neurons were studied in in vitro slice preparations. Type I neurons did not fire rhythmically but their spontaneous firing frequency ranged from 0 to 19.5 spikes/s (mean 9.7 spikes/s). The spontaneous firing of Type II neurons was rhythmic, with a mean frequency of 9.6 spikes/s (range 3.5-16.0 spikes/s). Choline acetyltransferase immunohistochemistry combined with biocytin labeling indicated that none of the Type I neurons were immunopositive to choline acetyltransferase, while 60% (42 of 69) of Type II neurons were immunopositive. There was no apparent difference in the electrophysiological membrane properties of immunopositive and immunonegative Type II neurons. At membrane potentials subthreshold for Na+ spikes (-50 mV), spontaneous membrane oscillations (11.6 Hz) were observed: these underlie the spontaneous repetitive firing of Type I neurons. The subthreshold membrane oscillation was tetrodotoxin sensitive but was not affected by Ca2+-free medium. A similar tetrodotoxin-sensitive subthreshold membrane oscillation (10.5 Hz) was also observed in Type II neurons. However, in Type II neurons a membrane oscillation was also observed at higher membrane potentials (-50 mV). This high-threshold oscillation was insensitive to tetrodotoxin and Na+-free medium, but was eliminated in Ca2+-free conditions. The amplitude and frequency of the high-threshold oscillation was increased upon membrane depolarization. At the most prominent oscillatory level (around -40 mV), the high-threshold oscillation had a mean frequency of 8.8 Hz. The high-threshold Ca2+ spike was triggered from the peak potential (-35 to -30mV) of the high-threshold oscillation. Application of tetraethylammonium chloride (< 5 mM) increased the amplitude of the high-threshold oscillation, while nifedipine greatly attenuated the high-threshold oscillation without changing the shape of the high-threshold Ca2+ spike. Application of Cd2+ eliminated both the high-threshold oscillation and the high-threshold Ca2+ spike, and omega-conotoxin reduced the size of the high-threshold Ca2+ spike without affecting the frequency of the high-threshold oscillation. Nickel did not have any effect on either the high-threshold oscillation or the high-threshold Ca2+ spike. These data suggest an involvement of N- and L-type Ca2+ channels in the generation of the high-threshold oscillation and the high-threshold Ca2+ spike, respectively. The results indicate that a persistent Na+ conductance plays a crucial role in the subthreshold membrane oscillation, which underlies spontaneous repetitive firing in Type I neurons. On the other hand, in addition to a persistent Na+ conductance for subthreshold membrane oscillation, a voltage-dependent Ca2+ conductance with Ca2+-dependent K+ conductance (for the high-threshold oscillation) may be responsible for rhythmic firing of Type II neurons.

The dorsal tegmentum of the pontomesencephalic junction of the rat--immunohistochemistry (choline acetyltransferase, tyrosine hydroxylase, substance P) and NADPH-diaphorase histochemistry in frontal and horizontal sections.

37 complete frontal and horizontal series of rat brain were studied to compare the distribution of choline acetyltransferase- (ChAT), tyrosine hydroxylase- (TH), substance P- (SP), calbindin D- (Calb) and NADPH-diaphorase (NADPH-d)-positive cells within the cytoarchitectonic borders of the latero-dorsal tegmental nucleus (L-D) and its neighbourhood. We found the same distribution, number and morphology of NADPH-d-positive cells and ChAT-positive cells. Rostrally, there are no borders between NADPH-d-positive cells of L-D and NADPH-d-positive cells of the lateral part of the dorsal raphe nucleus. Only a few TH-positive cells are intermingled with ChAT/NADPH-d-positive cells at the lateral border of L-D. TH-positive cells are larger or the same size as cholinergic neutrons. Locus coeruleus and its rostral part is full of TH-positive cells and their fibres run ventromedially towards L-D. Barrington's nucleus appears in double staining (ChAT and TH or NADPH-d and TH) as an empty area bordered by ChAT- or NADPH-d-positive cells of L-D and TH-positive fibres of the locus coeruleus. Some of these fibres run through the Barrington's nucleus. The shape and size of SP-positive neurons is the same as ChAT- and NADPH-d-positive neurons. SP-positive neurons are sparsely distributed in all parts of L-D, but there are only a few SP-positive cells in its medial part. About 50% of the ChAT- and NADPH-d-positive cells are also SP-positive. Results are expressed by figures in three representative frontal sections and one horizontal section through the dorsal mesopontine tegmentum.

Monoamine oxidase B (MAOB)-containing structures in MAOA-deficient transgenic mice.

Monoamine oxidase (MAO)-containing structures were studied for the first time in type A MAO (MAOA)-deficient transgenic mice (Tg8) derived from C3H strain, using MAO enzyme histochemistry. In this mutant line, MAOA activity was not detected in neurons of the locus coeruleus. In contrast, in their dorsal raphe neurons, we noted an intense activity of type B MAO (MAOB). Based on pharmacological MAOA suppression experiments employing a specific inhibitor (clorgyline), we confirmed that the localization of MAOB-positive structures are not different between Tg8 mutant and normal C3H line. Many of MAOB-positive structures which have not been described previously in the rat, cat and primates were described in this study. In the forebrain, MAOB-containing neurons were discriminated in the striatum, septal nuclei, major island of Calleja, diagonal band, medial forebrain bundle, ventral pallidum and amygdaloid nucleus. Stained neurons in the thalamus and hypothalamus were much more extensively distributed in the mouse than the rat. Pontine laterodorsal tegmental neurons showed MAOB activity. The present data suggest that serotonin, a preferential substrate for MAOA, can be oxidized by MAOB in MAOA-deficient Tg8 mice.