Brain-wide Mapping of Mono-synaptic Afferents to Different Cell Types in the Laterodorsal Tegmentum.

The laterodorsal tegmentum (LDT) is a brain structure involved in distinct behaviors including arousal, reward, and innate fear. How environmental stimuli and top-down control from high-order sensory and limbic cortical areas converge and coordinate in this region to modulate diverse behavioral outputs remains unclear. Using a modified rabies virus, we applied monosynaptic retrograde tracing to the whole brain to examine the LDT cell type specific upstream nuclei. The LDT received very strong midbrain and hindbrain afferents and moderate cortical and hypothalamic innervation but weak connections to the thalamus. The main projection neurons from cortical areas were restricted to the limbic lobe, including the ventral orbital cortex (VO), prelimbic, and cingulate cortices. Although different cell populations received qualitatively similar inputs, primarily via afferents from the periaqueductal gray area, superior colliculus, and the LDT itself, parvalbumin-positive (PV+) GABAergic cells received preferential projections from local LDT neurons. With regard to the different subtypes of GABAergic cells, a considerable number of nuclei, including those of the ventral tegmental area, central amygdaloid nucleus, and VO, made significantly greater inputs to somatostatin-positive cells than to PV+ cells. Diverse inputs to the LDT on a system-wide level were revealed.

A GABAergic tecto-tegmento-tectal pathway in pigeons.

Previous studies have demonstrated that the optic tecta of the left and right brain halves reciprocally inhibit each other in birds. In mammals, the superior colliculus receives inhibitory γ-aminobutyric acid (GABA)ergic input from the basal ganglia via both the ipsilateral and the contralateral substantia nigra pars reticulata (SNr). This contralateral SNr projection is important in intertectal inhibition. Because the basal ganglia are evolutionarily conserved, the tectal projections of the SNr may show a similar pattern in birds. Therefore, the SNr could be a relay station in an indirect tecto-tectal pathway constituting the neuronal substrate for the tecto-tectal inhibition. To test this hypothesis, we performed bilateral anterograde and retrograde tectal tracing combined with GABA immunohistochemistry in pigeons. Suprisingly, the SNr has only ipsilateral projections to the optic tectum, and these are non-GABAergic. Inhibitory GABAergic input to the contralateral optic tectum arises instead from a nearby tegmental region that receives input from the ipsilateral optic tectum. Thus, a disynaptic pathway exists that possibly constitutes the anatomical substrate for the inhibitory tecto-tectal interaction. This pathway likely plays an important role in attentional switches between the laterally placed eyes of birds. J. Comp. Neurol. 524:2886-2913, 2016. © 2016 Wiley Periodicals, Inc.

Electrophysiological changes in laterodorsal tegmental neurons associated with prenatal nicotine exposure: implications for heightened susceptibility to addict to drugs of abuse.

Prenatal nicotine exposure (PNE) is a risk factor for developing an addiction to nicotine at a later stage in life. Understanding the neurobiological changes in reward related circuitry induced by exposure to nicotine prenatally is vital if we are to combat the heightened addiction liability in these vulnerable individuals. The laterodorsal tegmental nucleus (LDT), which is comprised of cholinergic, GABAergic and glutamatergic neurons, is importantly involved in reward mediation via demonstrated excitatory projections to dopamine-containing ventral tegmental neurons. PNE could lead to alterations in LDT neurons that would be expected to alter responses to later-life nicotine exposure. To examine this issue, we monitored nicotine-induced responses of LDT neurons in brain slices of PNE and drug naive mice using calcium imaging and whole-cell patch clamping. Nicotine was found to induce rises in calcium in a smaller proportion of LDT cells in PNE mice aged 7-15 days and smaller rises in calcium in PNE animals from postnatal ages 11-21 days when compared with age-matched control animals. While inward currents induced by nicotine were not found to be different, nicotine did induce larger amplitude excitatory postsynaptic currents in PNE animals in the oldest age group when compared with amplitudes induced in similar-aged control animals. Immunohistochemically identified cholinergic LDT cells from PNE animals exhibited slower spike rise and decay slopes, which likely contributed to the wider action potential observed. Further, PNE was associated with a more negative action potential afterhyperpolarization in cholinergic cells. Interestingly, the changes found in these parameters in animals exposed prenatally to nicotine were age related, in that they were not apparent in animals from the oldest age group examined. Taken together, our data suggest that PNE induces changes in cholinergic LDT cells that would be expected to alter cellular excitability. As the changes are age related, these PNE-associated alterations could contribute differentially across ontogeny to nicotine-mediated reward and may contribute to the particular susceptibility of in utero nicotine exposed individuals to addict to nicotine upon nicotine exposure in the juvenile period.

Cholinergic neurons expressing neuromedin K receptor (NK3) in the basal forebrain of the rat: a double immunofluorescence study.

By using a double immunofluorescence method we have examined the distribution of cholinergic neurons expressing neuromedin K receptor (NK3) in the rat brain and spinal cord. The distribution of neuromedin K receptor-like immunoreactive neurons completely overlapped with that of choline acetyltransferase-positive neurons in certain regions of the basal forebrain, e.g. the medial septal nucleus, nucleus of the diagonal band of Broca, magnocellular preoptic nucleus and substantia innominata. Partially overlapping distributions of neuromedin K receptor-like immunoreactive and choline acetyltransferase-positive neurons were found in the basal nucleus of Meynert, globus pallidus, ventral pallidum of the forebrain, tegmental nuclei of the pons and dorsal motor nucleus of the vagus. Neurons showing both neuromedin K receptor-like and choline acetyltransferase immunoreactivities, however, were found predominantly in the medial septal nucleus, nucleus of the diagonal band of Broca and magnocellular preoptic nucleus of the basal forebrain: 66-80% of these choline acetyltransferase-positive neurons displayed neuromedin K receptor-like immunoreactivity. Neurons showing both neuromedin K receptor-like and choline acetyltransferase immunoreactivities were hardly detected in other aforementioned regions of the forebrain, brainstem and spinal cord. The present study has provided morphological evidence for direct physiological modulation or regulation of cholinergic neurons by tachykinins through the neuromedin K receptor in the basal forebrain of rats.

Age-related changes of neuronal counts in the human pedunculopontine nucleus.

Cholinergic neurons in the basal forebrain and the upper brainstem undergo changes during aging and in dementia of the Alzheimer type, Parkinson's disease and progressive supranuclear palsy. Little is known about the effect of age on neurons in the tegmental pedunculopontine nucleus. Cholinergic neurons revealed by choline acetyltransferase immunohistochemistry were quantified in the brains of 20 subjects who died without neurological disorder between 28 and 101 years of age. A U-shaped relationship between cell counts and age was found, namely, a decrease in counts between 28 and 70, a minimum between 80 and 91 years of age, and, in four subjects aged 98-101 years counts comparable to those of subjects having died between 28 and 65 years. The findings suggest that the loss of cholinergic pedunculopontine nucleus neurons is not linear. In centenarians age-related neuronal decrease in pedunculopontine nucleus neurons may be slower or the stock of pedunculopontine nucleus neurons greater than in subjects dying earlier.

Dopaminergic cell group A8 in the monkey: anatomical organization and projections to the striatum.

The first part of the study was a quantitative analysis of the distribution of A8 neurons compared with that of A9 and A10 neurons by means of tyrosine hydroxylase and calbindin-D(28K) immunohistochemistry and image analysis in monkeys. Then the striatal projection of A8 neurons was studied using retrograde and anterograde tracing methods. It was compared with that originating in cell groups A9 and A10 by performing injections of the retrograde tracer wheat germ agglutinin conjugated to horseradish peroxidase into different regions of the striatum. Ten percent of all mesencephalic dopaminergic neurons are located in cell group A8. This cell group, along with A10 and the dorsal part of A9, constitutes the dorsal tier, which accounts for 28% of mesencephalic dopaminergic neurons. Double-staining experiments showed that the neurons located in the dorsal tier were calbindin positive, whereas those from the ventral tier were not. In terms of anatomical projection, the dorsal tier mainly projects to the ventral part of the associative striatum, with preferential projections of A8 neurons to the ventrocaudal putamen, of A10 neurons to the nucleus accumbens, and of dorsal A9 neurons to both. Conversely, the main targets of the ventral tier of mesencephalic neurons (ventral part of A9) are the sensorimotor putamen and the associative caudate nucleus. In conclusion, each mesencephalic cell group projects primarily to one specific striatal region but also participates, albeit to a lesser extent, in the innervation of all the remaining striatal parts.

Localization of amino acids, neuropeptides and cholinergic neurotransmitter markers in identified projections from the mesencephalic tegmentum to the mammillary nuclei of the rat.

Retrograde labelling has been combined with immunohistochemistry to localize neurons containing GABA, glutamate, choline acetyltransferase, leu-enkephalin, neurotensin and substance P-like immunoreactivity in the projection pathways from the midbrain tegmental nuclei to the mammillary nuclei in the rat. Injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the medial mammillary nucleus resulted in retrogradely labelled neurons in the ventral tegmental nucleus of Gudden, whereas injections into the lateral mammillary nucleus resulted in large numbers of retrogradely labelled neurons in the ipsilateral dorsal tegmental nucleus of Gudden and in the laterodorsal tegmental nucleus. In the ventral tegmental nucleus, moderate to small numbers of retrogradely labelled neurons were also immunolabelled for GABA and approximately ten to 18 WGA-HRP-labelled neurons per section were immunoreactive for leu-enkephalin. In addition, small numbers of WGA-HRP-labelled neurons in the principal subnucleus of the ventral tegmental nucleus were immunoreactive for Glu whereas small numbers of retrogradely labelled neurons in the compact subnucleus of the central superior nucleus displayed neurotensin-like immunoreactivity. In the ventral subnucleus of the dorsal tegmental nucleus, moderate to small numbers of retrogradely labelled neurons were also GABA-immunoreactive and approximately ten to 14 WGA-HRP labelled neurons per section were immunoreactive for leu-enkephalin. The ventral subnucleus of the dorsal tegmental nucleus also contained small numbers of retrogradely labelled neurons that displayed either glutamate or substance P-like immunoreactivity. In addition, moderate to small numbers of WGA-HRP-labelled neurons (five to 20 per section) in the laterodorsal tegmental nucleus were immunoreactive for choline acetyltransferase. These results are compatible with the possibility that tegmentomammillary projection neurons use several different neurochemicals as neurotransmitter(s) and/or neuromodulator(s).

Calretinin-immunoreactive neurons in primate pedunculopontine and laterodorsal tegmental nuclei.

Single- and double-antigen localization procedures were used to study the distribution, morphological characteristics and chemical phenotype of neurons containing the calcium-binding protein calretinin in the pedunculopontine and laterodorsal tegmental nuclei of the cynomolgus monkey (Macaca fascicularis). Calretinin was detected in neurons that belonged to a highly heteromorphic and widely distributed subpopulation of the pedunculopontine and laterodorsal tegmental nuclei in the cynomolgus monkey. Double-immunostaining experiments revealed that about 12% of these calretinin-containing neurons displayed immunoreactivity for another calcium-binding protein, Calbindin-D28k. The calretinin/Calbindin-D28k double-labeled neurons had small to medium-sized perikarya, from which emerged a bipolar or multipolar dendritic arborization. Calretinin was also present in approximately 8% of the cholinergic neurons of the pedunculopontine/laterodorsal nuclear complex, as visualized on single sections immunostained for both calretinin and choline acetyltransferase. These calretinin/choline acetyltransferase double-labeled neurons displayed markedly different sizes and shapes, and occurred preferentially in the pars compacta and dissipata of the pedunculopontine tegmental nucleus. Numerous calretinin-immunoreactive fibers were also present within and around the superior cerebellar peduncle. Some of these varicose fibers closely surrounded large non-immunoreactive neurons, as well as large neurons staining positively for choline acetyltransferase. This study provides the first evidence for the existence of calretinin-immunoreactive neurons within the primate pedunculopontine and laterodorsal tegmental nuclei. Our data suggest that calretinin may play a role in the function of the pedunculopontine/laterodorsal nuclear complex by acting either alone or in conjunction with acetylcholine or Calbindin-D28k.

Differential expression of inflammatory mediators in rat microglia cultured from different brain regions.

Microglial cells show a rather uniform distribution of cell numbers throughout the brain with only minor prevalences in some brain regions. Their in situ morphologies, however, may vary markedly from elongated forms observed in apposition with neuronal fibers to spherical cell bodies with sometimes extremely elaborated branching. This heterogeneity gave rise to the hypothesis that these cells are differentially conditioned by their microenvironment and, therefore, also display specific patterns of differential gene expression. In this study, microglia were isolated from 2-4 week-old mixed CNS cultures that had been prepared from neonatal rat diencephalon, tegmentum, hippocampus, cerebellum and cerebral cortex, and were investigated 24 h later. Messenger RNA levels of proteins involved in crucial immune functions of this cell type (TNF-alpha, CD4, Fcgamma receptor II, and IL-3 receptor beta-subunit) have been determined by semi-quantitative RT-PCR. The results clearly show, that three of these mRNAs (TNF-alpha, CD4, Fcgamma receptor II) are differentially expressed in microglia with hippocampal microglia displaying the highest levels of these mRNAs. The data strongly support the notion that the status of microglial gene expression depends on their localization in brain and on specific interactions with other neural cell types. Consequently, it is hypothesized that their responsiveness to signals arising in injury or disease may vary from one brain region to another.

Evidence for co-existence of CCK-8 and GnRH in neurons of the mesencephalic tegmentum in the chameleon.

A double-label immunofluorescence technique was used to demonstrate the co-localization of cholecystokinin-8 (CCK-8) and gonadotrophin-releasing hormone (GnRH) in individual neurons and processes of the chameleon brain. Co-localization was limited to a small population of cells in the dorsomedial tegmentum; in other regions of the brain, neurons were observed to be either CCK-8-immunopositive or GnRH-immunopositive but never both. However, double-labeled fibers and terminals were found to be distributed at a low density throughout the thalamus, the medial hypothalamus, the tegmentum and the spinal cord. These data provide the first indication for the co-localization of CCK-8 and GnRH, whose functional significance remains to be established. ON

Hypothalamic and extrahypothalamic sauvagine-like immunoreactivity in the bullfrog (Rana catesbeiana) central nervous system.

In the present study, immunocytochemistry and radioimmunoassay were used to investigate the presence of sauvagine in both hypothalamic and extrahypothalamic areas of the central nervous system (CNS) of the bullfrog (Rana catesbeiana) using a specific antiserum raised against synthetic non-conjugated sauvagine (SVG), a frog (Phyllomedusa sauvagei) skin peptide of the corticotropin-releasing factor (CRF) family. Sauvagine-immunoreactive (SVG-ir) bipolar neurons were found in the nucleus of the fasciculus longitudinalis medialis located in the rostral mesencephalic tegmentum. In the tectal mesencephalon, beaded SVG-ir fibres were present in the optic tectum, and in the torus semicircularis. Abundant SVG-ir varicose fibres were seen in the granulosa layer of the cerebellum, the nucleus isthmi, and the obex of the spinal cord. SVG-ir fibres were also seen by the alar plate of the rombencephalon. In the diencephalon, the antiserum stained parvocellular neurons of the preoptic nucleus (PON) which extended their dendrites into the cerebro-spinal fluid (CSF) of the third ventricle and projected their ependymofugal fibres to the zona externa (ZE) of the median eminence. Immunopositive fibres were also present in the medial forebrain bundle at the chiasmatic field, the posterior thalamus, the pretectal gray, and the ventrocaudal hypothalamus. In the telencephalon (forebrain), SVG-ir fibres were seen in the medial septum, the lateral septum, and the amygdala. The SVG immunoreactivity could not be detected after using the SVG antiserum previously immunoabsorbed with synthetic SVG (0.1 microM), but immunoblock of the antiserum with sucker (Catostomus commersoni) urotensin I (sUI), sole (Hippoglossoides elassodon) urotensin I, sucker CRF, rat/human CRF, or ovine CRF (0.1-10 microM) did not eliminate visualization of the immunoreactivity. In radioimmunoassay, the SVG antiserum did not crossreact with sUI, or the SVG fragments SVG1-16, SVG16-27, and SVG26-34, but it recognized the C-terminal fragment SVG35-40. Crossreaction with mammalian ovine CRF and rat/human CRF was negligible. Both hypothalamic and mesencephalic extracts gave parallel displacement curves to SVG. The results suggest the presence in the bullfrog brain of a SVG-like neuropeptide, i.e., a peptide of the CRF family, that either is SVG or shares high homology with the C-terminus of that peptide. The function of this neuropeptide in amphibians is not known at this time, but based on its anatomical distribution to the ZE it could affect the release of adrenocorticotropin (ACTH) or other substances from the amphibian pars distalis. Involvement of the SVG-like peptide in behavioural (forebrain), visual (thalamus-tegmentum mesencephali-pretectal gray-optic tectum), motor coordination (cerebellum), and autonomic (spinal) functions, as well as an undefined interaction with the CSF in the bullfrog, seems likely.

Cytoarchitectural distribution of calcium binding proteins in midbrain dopaminergic regions of rats and humans.

The present study compares the distribution of three calcium binding proteins, calbindin-D28k, calretinin, and parvalbumin, in the midbrain tegmentum of rats and humans. In order to compare the distributions of these proteins directly, the cytoarchitecture of this region was evaluated by using immunohistochemistry for tyrosine hydroxylase and substance P in serial sections in both transverse and horizontal planes. There was a high degree of homology in the cytoarchitecture of the three main dopaminergic regions identified. The A8 group was localised in the retrorubral fields, which extended rostrally into the midbrain reticular fields in the human. The A9 group corresponded to the substantia nigra, which was delimited by its dense substance P innervation. The heterogeneous A10 group, situated along the dorsal border as well as medial to the A9 group, comprised multiple nuclei. The distribution of calcium binding proteins was similar in both species, although a larger proportion of neurons contained these proteins in the rat. Calbindin-D28k was localised in neurons within A8 and A10 nuclei and within the caudomedial A9 region (and rostrolateral A9 in the rat only). Calretinin was localised in similar regions. In contrast, neurons containing parvalbumin were concentrated in the substantia nigra pars reticulata. The results suggest that few dopaminergic neurons receiving striatal input in the substantia nigra contain calcium binding proteins; rather, the nondopaminergic nigral neurons contain parvalbumin. Interestingly, dopaminergic neurons are more numerous in humans, whereas nondopaminergic neurons predominate in rats, which suggests that functional differences may exist between rats and humans.

Difference in extracellular cocaine concentration between the ventral tegmental area and the medial prefrontal cortex following intravenous administration as revealed by quantitative microdialysis coupled with in vivo calibration.

Two microdialysis probes (active length: 1 mm) were simultaneously lowered into the ventral tegmental area (VTA) and the medial prefrontal cortex (mPFC) of each male, adult Sprague-Dawley rat, anesthetized with chloral hydrate, to evaluate whether cocaine distribution between these two areas is uniform after i.v. injection of 3 mg/kg cocaine. Using a modified in vivo calibration method, we can estimate the extracellular cocaine concentration in those nuclei. This proposed calibration method offers the advantages of simplicity and speed. Measurement of the cocaine concentration in the dialysate and estimation of the extracellular cocaine concentration showed that extracellular cocaine in the VTA was significantly higher than that in the mPFC. Next, the active length of the dialysis probe was extended to 4 mm in order to examine the effect of the active probe length on the cocaine distribution profile between the mPFC and a mixed area of VTA and red nucleus. Surprisingly, the cocaine concentration in dialysate had a contradictory result compared to the extracellular cocaine concentration due to in vivo recovery in the mPFC (25.3 +/- 3.1%) which was significantly higher than that in the mixed area (17.5 +/- 1.6%). Firstly, these results suggest that distribution of cocaine following systemic administration was not uniform in the rat brain. Secondly, in a valid comparison of drug concentrations between the two dialysis areas, we must consider the in vivo recovery for each brain region.

Intrastriatal implantation of interleukin-1. Reduction of parkinsonism in rats by enhancing neuronal sprouting from residual dopaminergic neurons in the ventral tegmental area of the midbrain.

Intrastriatal implantation with dopaminergic of nondopaminergic tissue can elicit behavioral recovery in parkinsonian animals. Because in these animals, especially in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned monkeys, there are still considerable numbers of dopaminergic neurons left in the mesencephalon, implantation-induced trophic effects on host residual dopaminergic neurons have been suggested as a mechanism underlying the behavioral recovery. Gliosis around the graft is a universal finding in any implantation procedure and is probably mediated by interleukin-1 (IL-1); in addition, activated astrocytes secrete several neurotrophic factors in vitro. Therefore, the authors postulated that trophic effects from IL-1-induced gliosis may be a "final common pathway" for recovery in parkinsonian animals after implantation. Hemiparkinsonism was induced in rats by injection of 6-hydroxydopamine either directly into the substantia nigra or into the median forebrain bundle. The substantia nigra-lesioned rats showed complete depletion of dopaminergic neurons in the substantia nigra but sparing of those in the ventral tegmental area, whereas the median forebrain bundle-lesioned animals had depletion of dopaminergic cells in the substantia nigra and the ventral tegmental area. Polymer pellets containing either slow-released IL-1 alpha and beta or placebo pellets were implanted in the caudate nucleus on the lesioned side in both groups. The rats' rotational response to amphetamine was tested weekly for 8 weeks. Selective substantia nigra-lesioned rats with implantation of IL-1 pellets had a 45% reduction in amphetamine-induced rotation, whereas placebo-implanted substantia nigra-lesioned rats had a 14% reduction in rotation. In the median forebrain bundle-lesioned group, neither IL-1 nor placebo implantation elicited any effect on turning. Immunohistochemical staining for glial fibrillary acidic protein was markedly increased surrounding the IL-1 pellets compared to the placebo pellets. In the selective substantia nigra-lesioned rats with IL-1 pellets implanted in the caudate nucleus, a considerable number of tyrosine hydroxylase immunoreactive (TH-IR) fibers were observed in the medial and middle portions of the caudate nucleus. Fewer TH-IR fibers were seen in the rats with placebo-bearing pellets. These results suggest that neurotrophic activities mediated by IL-1 and reactive astrocytes might be a common path through which tissue trauma and some tissue transplants exert their beneficial effects in parkinsonian animals. Furthermore, most of the sprouted dopaminergic fibers induced by IL-1 in the caudate nucleus come from dopaminergic neurons in the ventral tegmental area.

5-HT2 receptor immunoreactivity on cholinergic neurons of the pontomesencephalic tegmentum shown by double immunofluorescence.

The serotonin-2 (5-HT2) receptor subtype is implicated in several behavioral and physiological processes, and may be the site of action of hallucinogens and certain psychotherapeutic drugs. To better understand the function and regulation of 5-HT2 receptors, it is necessary to determine the specific brain regions and cell types expressing them. By double immunofluorescence using a polyclonal antibody raised against the rat 5-HT2 receptor in conjunction with an antibody against choline acetyltransferase (ChAT), the synthetic enzyme for acetylcholine, we have shown that cholinergic neurons in the rat laterodorsal and pedunculopontine tegmental nuclei express 5-HT2 receptors. In contrast, there was little co-localization of 5-HT2 and ChAT immunoreactivity in neurons of the basal forebrain or striatum, even though the 5-HT2- and ChAT-positive cells in these regions overlapped extensively. These findings are discussed in relation to the potential interaction between cholinergic and serotonergic systems in sleep regulation, hallucinogenesis and the pathophysiology of neuropsychiatric disorders.

Opioid receptors in midbrain dopaminergic regions of the rat. II. Kappa and delta receptor autoradiography.

Opiates and opioid peptides are known to influence the dopaminergic (DA) neurons in the midbrain. The purpose of this study was to map and quantify the density of kappa and delta opioid receptor subtypes in the retrorubral field, substantia nigra, and ventral tegmental area and related nuclei, which contain DA nuclei A8, A9, and A10, respectively. Sections through the rostral-caudal extent of the rat midbrain were stained with an antibody against tyrosine hydroxylase, as a DA cell marker, and comparable sections were processed for in vitro receptor autoradiography using the kappa-selective ligand, U-69593, and the delta-selective ligand, D-Pen2, D-Pen5-enkephalin. In general, both kappa and delta ligands exhibited low levels of specific binding in regions occupied by the midbrain DA neurons. Kappa binding (4-8 fmol/mg tissue) was high throughout the rostral-caudal extent of the substantia nigra, in rostral portions of the ventral tegmental area, and in the nucleus paranigralis; low binding occurred in the retrorubral field and central linear nucleus raphe. Delta binding (6-18 fmol/mg tissue) was high in the caudal portion of the substantia nigra pars reticulata, and in the medial terminal nucleus of the accessory optic system (a region previously shown to contain DA dendrites). The kappa and delta receptor binding is heterogeneously distributed in regions occupied by midbrain dopaminergic neurons, and several fold lower than the binding of mu opioid receptors in the same brain regions.

Opioid receptors in midbrain dopaminergic regions of the rat. I. Mu receptor autoradiography.

Several lines of evidence indicate that an interaction exists between opioid peptides and midbrain dopaminergic neurons. The purpose of this study was to map and quantify the density of the mu opioid receptor subtype relative to the location of the dopaminergic (DA) neurons in the retrorubral field (nucleus A8), substantia nigra (nucleus A9), and ventral tegmental area and related nuclei (nucleus A10) in the rat. Sections through the rostral-caudal extent of the midbrain were stained with an antibody against tyrosine hydroxylase, as a DA cell marker, and comparable sections were processed for in vitro receptor autoradiography using the mu-selective ligand, 3H-Tyr-D-Ala-N-MePhe-Gyl-ol enkephalin. In the nucleus A8 region, there were low levels of mu binding. In the rostral portion of nucleus A9, there was prominent mu binding both in the ventral pars compacta, which contains numerous DA neurons, and in regions that correspond to the location of the DA dendrites which project ventrally into the underlying substantia nigra pars reticulata. In the caudal portion of nucleus A9, mu binding was greatest in the substantia nigra pars reticulata, but also in the same region that contains DA neurons. In nucleus A10, mu receptor densities differed depending upon the nucleus A10 subdivision, and the rostral-caudal position in the nucleus. Low receptor densities were observed in rostral portions of the ventral tegmental area and interfascicular nucleus, and there was negligible binding in the parabrachial pigmented nucleus and paranigral nucleus at the level of the interpeduncular nucleus; all regions where there are high densities of DA somata. Mu binding was relatively high in the central linear nucleus, and in the dorsal and medial divisions of the medial terminal nucleus of the accessory optic system, which has been shown to contain DA dendrites. These data indicate that mu opioid receptors are located in certain regions occupied by all three midbrain DA nuclei, but in a highly heterogeneous fashion.

Development of tyrosine hydroxylase-, dopamine- and dopamine beta-hydroxylase-immunoreactive neurons in a teleost, the three-spined stickleback.

The development of catecholaminergic neuronal systems in the brain of a teleost, the three-spined stickleback, was studied through embryonic to early larval stages by immunocytochemistry using specific antibodies against dopamine, tyrosine hydroxylase and dopamine beta-hydroxylase. By analysing the spatiotemporal patterns of development for the catecholaminergic nuclei, possible homologies with nuclei in amniote brains have been identified. The noradrenergic neurons in the isthmus region of the rostral rhombencephalon originate in the same manner as the A4-A7 + subcoeruleus group in mammals. Their developmental characteristics show the largest similarities with the subcoeruleus group of birds and mammals, although some features are shared with developing A6 (locus coeruleus) neurons. Catecholaminergic neurons never appear during development in the ventral mesencephalon of the three-spined stickleback. A group of large dopaminergic neurons that accompany the cerebrospinal fluid (CSF)-contacting neurons follows the border between the hypothalamus and the ventral thalamus into the caudal hypothalamus, where they are continuous with the dopaminergic neurons in the posterior tuberculum. They are thus topologically comparable with the dopaminergic neurons of the zona incerta in mammals. The dopaminergic CSF-contacting neurons that line the median, lateral and posterior recesses of the third ventricle do not contain tyrosine hydroxylase-immunoreactivity at any developmental stage. This indicates that they take up and accumulate exogenous dopamine or L-dihydroxyphenylalanine, and do not synthesize dopamine from tyrosine at any developmental stage. Tyrosine hydroxylase-immunoreactive neurons appear in the pineal organ on the day of hatching (120 h post-fertilization). They were still observed in 240-h-old larvae, but are absent in the pineal organ of adult sticklebacks. The initial appearance and subsequent differentiation of catecholaminergic neurons in the stickleback embryo follow essentially the same spatial and temporal pattern as in amphibian, avian and mammalian embryos. This observation supports the hypothesis that morphologically, topologically and chemically similar monoaminergic neurons in different vertebrate classes are homologous.

Further data on the development of SRIF-like immunoreactive nerve cell populations in the chick embryo brain stem. II. Midbrain tegmentum.

Further immunocytochemical analysis of the neuroblasts with SRIF-like immunoreactivity (ir) was carried out on the chick embryo midbrain tegmentum. 5 or 100 microns mesencephalon sections were obtained from 60 White Leghorn chick embryos at stages (E = Embryonic days) ranging from E4 1/2 to E18 and incubated with rabbit polyclonal antibodies against synthetic cyclic Somatostatin-14, according to PAP-DAB technique. In the midbrain tegmentum the ir appeared as from E12. From E12 to E13 1/2-E14 the ir distribution gradually changed. From E14 to E18 numbers and spatial arrangement of the positive neuroblast groups did not show substantial changes; in these respects the ir distributional pattern proved to be similar to the one observed by the Authors in adult animals. From E17 to E18 a decrease in the positive neuroblast density appeared to occur, particularly in a ventrally placed group. These results are consistent with a possible local regulative role of the SRIF.

Substance P-containing neurons in the mesopontine tegmentum are severely affected in Parkinson's disease.

Substance P immunoreactive (SP+) neurons were analysed quantitatively in serial sections of the mesopontine tegmentum in 6 patients with idiopathic Parkinson's disease and 5 age-matched normal controls. In the tegmentum of the Parkinson's disease brains many SP+ neurons contained swollen, twisted neuronal processes as well as Lewy bodies. There were significant reductions in the total number of SP+ neurons in the pedunculopontine tegmental nucleus (loss 43%), in the laterodorsal tegmental nucleus (loss 28%), in the oral pontine reticular nucleus (loss 41%) and in the median raphe nucleus (loss 76%). It was the large SP+ (greater than 20 microns) neurons that were particularly affected. In our control group we did not document a significant relationship between age at death and number of SP+ neurons in these tegmental nuclei or between age at death and number of pigmented neurons in the locus coeruleus. In contrast, in patients with Parkinson's disease, there was a strong inverse relationship between age at death and numbers of SP+ and pigmented neurons. Our findings suggest an interaction between the pathophysiological mechanisms initiated by Parkinson's disease and other processes related to ageing. Since tegmental SP+ neurons are affected by the primary pathological processes underlying Parkinson's disease as severely as catecholamine-synthesizing neurons are affected, theories of pathogenesis and therapeutic strategies in Parkinson's disease will need to take into account the involvement of these SP+ neurons.