Development of glycine immunoreactivity in the brain of the sea lamprey: Comparison with γ‐aminobutyric acid immunoreactivity

The development of glycine immunoreactivity in the brain of the sea lamprey was studied by use of immunofluorescence techniques at embryonic to larval stages. Glycine distribution was also compared with that of γ‐aminobutyric acid (GABA) by use of double immunofluorescence. The first glycine‐immunoreactive (ir) cells appeared in the caudal rhombencephalon of late embryos, diencephalon of early prolarvae, and mesencephalon of late prolarvae, in which glycine‐ir cells were observed in several prosencephalic regions (preoptic nucleus, hypothalamus, ventral thalamus, dorsal thalamus, pretectum, and nucleus of the medial longitudinal fascicle), mesencephalon (M5), isthmus, and rhombencephalon. In larvae, glycine‐ir populations were observed in the olfactory bulbs, preoptic nucleus and thalamus (prosencephalon), M5 and oculomotor nucleus (mesencephalon), dorsal isthmic gray, isthmic reticular formation, and various alar and basal plate rhombencephalic populations. No glycine‐ir cells were observed in the larval optic tectum or torus semicircularis, which contain glycine‐ir populations in adults. A wide distribution of glycine‐ir fibers was observed, which suggests involvement of glycine in the function of most lamprey brain regions. Colocalization of GABA and glycine in prolarvae was found mainly in cell groups of the diencephalon, in the ventral isthmic group, and in trigeminal populations. In larvae, colocalization of GABA and glycine was principally observed in the M5 nucleus, the reticular formation, and the dorsal column nucleus. The present results reveal for the first time the complex developmental pattern of the glycinergic system in lamprey, including early glycine‐ir populations, populations transiently expressing glycine, and late‐appearing populations, in relation to maturation changes that occur during metamorphosis. J. Comp. Neurol. 512:747–767, 2009. © 2008 Wiley‐Liss, Inc.     

Immunohistochemical distribution of calretinin and calbindin (D‐28k) in the brain of the cladistian Polypterus senegalus

Polypteriform fishes are believed to be basal to other living ray‐finned bony fishes, and they may be useful for providing information of the neural organization that existed in the brain of the earliest ray‐finned fishes. The calcium‐binding proteins calretinin (CR) and calbindin‐D28k (CB) have been widely used to characterize neuronal populations in vertebrate brains. Here, the distribution of the immunoreactivity against CR and CB was investigated in the olfactory organ and brain of Polypterus senegalus and compared to the distribution of these molecules in other ray‐finned fishes. In general, CB‐immunoreactive (ir) neurons were less abundant than CR‐ir cells. CR immunohistochemistry revealed segregation of CR‐ir olfactory receptor neurons in the olfactory mucosa and their bulbar projections. Our results confirmed important differences between pallial regions in terms of CR immunoreactivity of cell populations and afferent fibers. In the habenula, these calcium‐binding proteins revealed right‐left asymmetry of habenular subpopulations and segregation of their interpeduncular projections. CR immunohistochemistry distinguished among some thalamic, pretectal, and posterior tubercle‐derived populations. Abundant CR‐ir populations were observed in the midbrain, including the tectum. CR immunoreactivity was also useful for characterizing a putative secondary gustatory/visceral nucleus in the isthmus, and for distinguishing territories in the primary viscerosensory column and octavolateral region. Comparison of the data obtained within a segmental neuromeric context indicates that some CB‐ir and CR‐ir populations in polypteriform fishes are shared with other ray‐finned fishes, but other positive structures appear to have evolved following the separation between polypterids and other ray‐finned fishes. J. Comp. Neurol. 521:2454–2485, 2013. © 2013 Wiley Periodicals, Inc.     

Cholinergic profiles in the Goettingen miniature pig (Sus scrofa domesticus) brain

Central cholinergic structures within the brain of the even‐toed hoofed Goettingen miniature domestic pig (Sus scrofa domesticus) were evaluated by immunohistochemical visualization of choline acetyltransferase (ChAT) and the low‐affinity neurotrophin receptor, p75^NTR. ChAT‐immunoreactive (‐ir) perikarya were seen in the olfactory tubercle, striatum, medial septal nucleus, vertical and horizontal limbs of the diagonal band of Broca, and the nucleus basalis of Meynert, medial habenular nucleus, zona incerta, neurosecretory arcuate nucleus, cranial motor nuclei III and IV, Edinger‐Westphal nucleus, parabigeminal nucleus, pedunculopontine nucleus, and laterodorsal tegmental nucleus. Cholinergic ChAT‐ir neurons were also found within transitional cortical areas (insular, cingulate, and piriform cortices) and hippocampus proper. ChAT‐ir fibers were seen throughout the dentate gyrus and hippocampus, in the mediodorsal, laterodorsal, anteroventral, and parateanial thalamic nuclei, the fasciculus retroflexus of Meynert, basolateral and basomedial amygdaloid nuclei, anterior pretectal and interpeduncular nuclei, as well as select laminae of the superior colliculus. Double immunofluorescence demonstrated that virtually all ChAT‐ir basal forebrain neurons were also p75^NTR‐positive. The present findings indicate that the central cholinergic system in the miniature pig is similar to other mammalian species. Therefore, the miniature pig may be an appropriate animal model for preclinical studies of neurodegenerative diseases where the cholinergic system is compromised. J. Comp. Neurol. 525:553–573, 2017. © 2016 Wiley Periodicals, Inc.     

Distribution of vasoactive intestinal peptide-like immunoreactivity in the brain and pituitary of the frog (Rana esculenta) during development

The localization of vasoactive intestinal peptide (VIP)-like immunoreactive (ir) elements was investigated in the brain of the anuran amphibian, Rana esculenta, during development. Using an antiserum raised against the porcine VIP, ir cell bodies and fibers were observed in the forebrain of tadpoles a few days after hatching. During early premetamorphosis, ir perikarya were distributed in the ventral infundibular nucleus of the hypothalamus and in the posterocentral nucleus of the thalamus. Labeled fibers were detected in the olfactory bulbs and in the hypothalamus. In these larvae, furthermore, several VIP-ir cells were found in the pars distalis of the pituitary and there were ir fibers in the pars nervosa. In tadpoles at stages VIII-IX, a new group of VIP-labeled neurons was observed in the dorsal part of the infundibular nucleus. In other brain regions, the distribution of the immunoreactivity was similar to that described in the earliest stages, i.e., IV-VII. During mid-premetamorphosis, stages X-XII of development, an additional set of ir perikarya appeared in the ventrolateral area of the thalamus. During late premetamorphosis, stages XIII-XVIII, the organization of VIP-like immunoreactivity was more complex and its distribution more widespread. Two new groups of ir cell bodies appeared, one in the preoptic nucleus and another in the anteroventral area of the thalamus, and for the first time, VIP immunoreactivity was observed in the median eminence. This distribution pattern persisted through to the prometamorphic, four-limb stage. Strikingly, no VIP-ir elements were observed anywhere in the mid- and hindbrain. The present results indicate that a VIP-like ir peptide may be involved in the processing of olfactory information or may act as a neurohormone, hypophysiotropic factor, and neuromodulator in the brain of R. esculenta during development.     

Somatostatin interneurons delineate the inner part of the external plexiform layer in the mouse main olfactory bulb

Neuropeptides play a major role in the modulation of information processing in neural networks. Somatostatin, one of the most concentrated neuropeptides in the brain, is found in many sensory systems including the olfactory pathway. However, its cellular distribution in the mouse main olfactory bulb (MOB) is yet to be characterized. Here we show that ≈95% of mouse bulbar somatostatin‐immunoreactive (SRIF‐ir) cells describe a homogeneous population of interneurons. These are restricted to the inner lamina of the external plexiform layer (iEPL) with dendritic field strictly confined to the region. iEPL SRIF‐ir neurons share some morphological features of Van Gehuchten short‐axon cells, and always express glutamic acid decarboxylase, calretinin, and vasoactive intestinal peptide. One‐half of SRIF‐ir neurons are parvalbumin‐ir, revealing an atypical neurochemical profile when compared to SRIF‐ir interneurons of other forebrain regions such as cortex or hippocampus. Somatostatin is also present in fibers and in a few sparse presumptive deep short‐axon cells in the granule cell layer (GCL), which were previously reported in other mammalian species. The spatial distribution of somatostatin interneurons in the MOB iEPL clearly outlines the region where lateral dendrites of mitral cells interact with GCL inhibitory interneurons through dendrodendritic reciprocal synapses. Symmetrical and asymmetrical synaptic contacts occur between SRIF‐ir dendrites and mitral cell dendrites. Such restricted localization of somatostatin interneurons and connectivity in the bulbar synaptic network strongly suggest that the peptide plays a functional role in the modulation of olfactory processing. J. Comp. Neurol. 518:1976–1994, 2010. © 2010 Wiley‐Liss, Inc.     

Characterization of neuropeptide Y2 receptor protein expression in the mouse brain. II. Coexistence with NPY,the Y1 receptor,and other neurotransmitter‐related molecules

Neuropeptide Y (NPY) is widely expressed in the brain and its biological effects are mediated through a variety of receptors. We examined, using immunohistochemistry, expression of the Y2 receptor (R) protein in the adult mouse brain and its association with NPY and the Y1R, as well as a range of additional neurotransmitters and signaling‐related molecules, which previously has not been defined. Our main focus was on the hippocampal formation (HiFo), amygdaloid complex, and hypothalamus, considering the known functions of NPY and the wide expression of NPY, Y1R, and Y2R in these regions. Y2R‐like immunoreactivity (‐LI) was distributed in nerve fibers/terminal endings throughout the brain axis, without apparent colocalization with NPY or the Y1R. Occasional coexistence between NPY‐ and Y1R‐LI was found in the HiFo. Following colchicine treatment, Y2R‐LI accumulated in cell bodies that coexpressed γ‐aminobutyric acid (GABA) in a population of cells in the amygdaloid complex and lateral septal nucleus, but not in the HiFo. Instead, Y2R‐positive nerve terminals appeared to surround GABA‐immunoreactive (ir) cells in the HiFo and other neuronal populations, e.g., NPY‐ir cells in HiFo and tyrosine hydroxylase‐ir cells in the hypothalamus. In the HiFo, Y2R‐ir mossy fibers coexpressed GABA, glutamic acid decarboxylase 67 and calbindin, and Y2R‐LI was found in the same fibers that contained the presynaptic metabotropic glutamate receptor 2, but not together with any of the three vesicular glutamate transporters. Our findings provide further support that Y2R is mostly presynaptic, and that Y2Rs thus have a modulatory role in mediating presynaptic neurotransmitter release. J. Comp. Neurol. 519:1219–1257, 2011. © 2011 Wiley‐Liss, Inc.     

Projection from the accommodation-related area in the superior colliculus of the cat

Our previous study has indicated that accommodative responses can be evoked with weak currents applied to a circumscribed area of the superior colliculus in the cat. We investigated efferent projections from this area with biocytin in the present study. The accommodation area in the superior colliculus was identified by systematic microstimulation in each of five anesthetized cats. Accommodative responses were detected by an infrared optometer. After mapping the superior colliculus, biocytin was injected through a glass micropipette into the accommodation area, where accommodative responses were elicited with low-intensity microstimulation. In addition, accommodative responses to stimulation of the superior colliculus were compared before and after an injection of muscimol, an agonist of inhibitory neurotransmitter, into the pretectum. Following the injection of biocytin, in the ascending projections, labeled terminals were seen mainly in the caudal portion of the nucleus of the optic tract, the nucleus of the posterior commissure, the posterior pretectal nucleus, the olivary pretectal nucleus, the mesencephalic reticular formation at the level of the oculomotor nucleus, and the lateral posterior nucleus of the thalamus on the ipsilateral side. Less dense terminals were seen in the anterior pretectal nucleus, the zona incerta, and the centromedian nucleus of the thalamus. In the descending projections, labeled terminals were observed mainly in the paramedian pontine reticular formation, the nucleus raphe interpositus, and the dorsomedial portion of the nucleus reticularis tegmenti pontis on the contralateral side. Less dense terminals were also seen in the nucleus of the brachium of the inferior colliculus, the cuneiform nucleus, the medial part of the paralemniscal tegmental field, and the dorsolateral division of the pontine nuclei on the ipsilateral side. Following the injection of muscimol into the pretectum, including the nucleus of the optic tract, the posterior pretectal nucleus, and the nucleus of the posterior commissure, accommodative responses evoked by microstimulation of the superior colliculus were reduced to 33–55% of the value before the injections. These findings suggest that the accommodation area in the superior colliculus projects to the oculomotor nucleus through the ipsilateral pretectal area, especially the nucleus of the optic tract, the nucleus of posterior commissure, and the posterior pretectal nucleus, and also projects to the pupilloconstriction area (the olivary pretectal nucleus), the vergence-related area (the mesencephalic reticular formation), and the active visual fixation-related area (the nucleus raphe interpositus). © 1996 Wiley-Liss, Inc.     

Mapping of neuropeptide Y-like immunoreactivity in the human forebrain

By means of immunohistochemistry the distribution of neuropeptide Y (NPY)-containing neurons and fibres was determined in the human forebrain on the basis of frontal paraffin sections from six individuals of different biological age. NPY was located in abundance in telencephalic cortical and subcortical structures like the striatum, the amygdaloid body and the substantia innominata. Variations in the distribution of immunoreactivity were observed and correlated with distinct subdivisions and structural elements of the basal forebrain region identified by Weigert or Nissl-stained neighbouring sections. The diencephalon was characterized by a relative paucity of labeled cells which were mostly confined to the area of the Nc. infundibularis and the median eminence while fibers were widely distributed in high density in most hypothalamic subnuclei except for the supraoptic nucleus. A periventricular zone of high fibre immunoreactivity was observed in the thalamus. NPY distribution in the developing brain was characterized by the finding of numerous labeled perikarya in the subcortical white matter and by far higher densities of labeled cells in the striatum as compared to the adult brain.     

Gonadotropin‐inhibitory hormone identification,cDNA cloning,and distribution in rhesus macaque brain

Gonadotropin‐inhibitory hormone (GnIH) is a hypothalamic neuropeptide that modulates the reproductive physiology of birds and mammals by inhibiting gonadotropin secretion from the anterior pituitary gland. GnIH can also directly inhibit reproductive behaviors, possibly via action within the brain. Identification of the distribution of GnIH neurons and fibers may provide us with clues to how the brain controls reproductive activities of the animal. Here, we characterized the location and connectivity of GnIH neurons in the rhesus macaque (Macaca mulatta) brain. We determined the macaque GnIH precursor mRNA, and further identified a mature GnIH peptide (SGRNMEVSLVRQVLNLPQRF‐NH₂) by mass spectrometry combined with immunoaffinity purification. The majority of GnIH precursor mRNA‐positive and GnIH‐immunoreactive (GnIH‐ir) cell bodies were localized in the intermediate periventricular nucleus (IPe) in the hypothalamus, as determined by in situ hybridization and immunocytochemistry, respectively. Abundant GnIH‐ir fibers were observed in the nucleus of the stria terminalis in the telencephalon; habenular nucleus, paraventricular nucleus of the thalamus, preoptic area, paraventricular nucleus of the hypothalamus, IPe, arcuate nucleus of hypothalamus, median eminence and dorsal hypothalamic area in the diencephalon; medial region of the superior colliculus, central gray substance of the midbrain and dorsal raphe nucleus in the midbrain; and parabrachial nucleus in the pons. GnIH‐ir fibers were observed in close proximity to gonadotropin‐releasing hormone‐I, dopamine, β‐endorphin, and gonadotropin‐releasing hormone‐II neurons in the preoptic area, IPe, arcuate nucleus of hypothalamus, and central gray substance of midbrain, respectively. GnIH neurons might thus regulate several neural systems in addition to pituitary gonadotropin release. J. Comp. Neurol. 517:841–855, 2009. © 2009 Wiley‐Liss, Inc.     

The distribution of glutamic acid decarboxylase immunoreactivity in the diencephalon of the opossum and rabbit

We have examined the distribution of neurons and terminals immunoreactive for glutamic acid decarboxylase (GAD) in the thalamus and adjacent structures of the opossum (Didelphis virginiana) and the rabbit and have compared this distribution with the distributions we described previously for the cat and bushbaby (Galago senegalensis). The significance of these experiments depends, first, on the fact that GAD is the synthetic enzyme for GABA, and therefore that GAD immunoreactivity is a marker for GABAergic inhibitory neurons, and second, on previous findings that suggest that GABAergic neurons in the dorsal thalamus are local circuit neurons. In both cat and Galago, GAD-immunoreactive neurons are distributed essentially throughout the entire thalamus. In the opossum, GAD neurons are chiefly confined to the dorsal lateral geniculate nucleus and the lateral extremity of the lateral posterior nucleus. The distribution of GAD neurons in the rabbit is intermediate between that found in the opossum on the one hand and cat and Galago on the other. Like opossum, about 25% of the neurons in the lateral geniculate nucleus of rabbit are GAD immunoreactive. Unlike opossum, however, as many as 18% of the cells in the ventral posterior nucleus of the rabbit are GAD immunoreactive, and scattered cells are also labeled in other thalamic areas, such as the medial geniculate and the lateral group. Aside from the findings in the dorsal thalamus, the chief observation is that GAD-immunoreactive neurons and/or terminals densely fill all principal targets of the optic tract, including the ventral lateral geniculate nucleus; the superficial gray layer of the superior colliculus; the anterior, posterior, and olivary pretectal nuclei; the nucleus of the optic tract; and the medial and lateral terminal nuclei of the accessory optic tract. These results support the idea first put forward by Cajal that local circuit neurons increase in number during the course of the evolution of complex mammalian brains. If we can assume that the conservative opossum retains characteristics reflecting an early stage of mammalian evolution, the results suggest that thalamic local circuit neurons arose first in the visual system and only later in evolution spread throughout the thalamus.     

Effect of optic nerve transection onN-acetylaspartylglutamate immunoreactivity in the primary and accessory optic projection systems in the rat

Evidence has been presented in recent years that support the hypothesis thatN-acetylaspartylglutamate (NAAG) may be involved in synaptic transmission in the optic tract of mammals. Using a modified fixation protocol, we have determined the detailed distribution of NAAG immunoreactivity (NAAG-IR) in retinal ganglion cells and optic projections of the rat. Following optic nerve transection, dramatic losses of NAAG-IR were observed in the neuropil of all retinal target zones including the lateral geniculate nucleus, superior colliculus, nucleus of the optic tract, the dorsal and medial terminal nuclei and suprachiasmatic nucleus. Brain regions were microdissected and NAAG levels measured by a radioimmunoassay (RIA) (IC₅₀:NAAG= 2.5nM,NAA= 100 μM;smallest detectable amount= 1–2pg/assay). decreases (50–60%) in NAAG levels were detected in the lateral geniculate, superior colliculus and suprachiasmatic nucleus. Moderate losses (25–45%) were noted in the pretectal nucleus and the nucleus of the optic tract. Smaller changes (15–20%) were detected in the paraventricular nucleus and the pretectal area. These results are consistent with a synaptic communication role for NAAG in the visual system.     

Efferent connections from the anterior pretectal nucleus to the diencephalon and mesencephalon in the rat

The anterior pretectal nucleus has been described as part of the visual pretectal complex. However, several electrophysiological and behavioural studies showed that this area is involved in somatosensory modulation, more specifically, antinociception. The efferents of the anterior pretectal nucleus have not been identified taking into account the different function of this nucleus in relation to the rest of the pretectal complex. In the study herein described, a sensitive anterograde tracer Phaseolus vulgaris leucoagglutinin was used to trace the mesencephalic and diencephalic efferents of the anterior pretectal nucleus in the rat. The majority of the connections were ipsilateral. Fibres with varicosities were observed in discrete areas of the thalamus (central lateral, posterior complex), hypothalamus (lateral, posterior and ventromedial), zona incerta, parvocellular red nucleus, intermediate and deep layers of the superior colliculus, central grey, deep mesencephalon, pontine parabrachial region, and pontine nuclei. Fibres en passant were detected in the medial lemniscus, from the level of the injection site to rostral medullary levels. Some labelled axons were seen coursing to the contralateral side through the posterior commissure and the decussation of the superior cerebellar peduncle. These results show that the anterior pretectal nucleus projects principally to areas involved in somatosensory and motor control in a manner that permits sensory modulation at higher and lower levels of the brain. These connections may explain the antinociceptive and antiaversive effects of stimulating the anterior pretectal nucleus in freely moving animals.     

Sources of subcortical projections to the superior colliculus in the cat

A comprehensive search for subcortical projections to the cat superior colliculus was conducted using the retrograde horseradish peroxidase (HRP) method. Over 40 different subcortical structures project to the superior colliculus. The more notable among these are grouped under the following categories. Visual structures: ventral lateral geniculate nucleus, parabigeminal nucleus, pretectal area (nucleus of the optic tract, posterior pretectal nucleus, nuclei of the posterior commissure). Auditory structures: inferior colliculus (external and pericentral nuclei), dorsomedial periolivary nucleus, nuclei of the trapezoid body, ventral nucleus of the lateral lemniscus. Somatosensory structures: sensory trigeminal complex (all divisions, but mainly the γ division of nucleus oralis), dorsal column nuclei (mostly cuneate nucleus), and the lateral cervical nucleus. Catecholamine nuclei: locus coeruleus, raphe dorsalis, and the parabrachial nuclei. Cerebellum: medial, interposed, and lateral nuclei, and the perihypoglossal nuclei. Reticular areas: zona incerta, substantia nigra, midbrain tegmentum, nucleus paragigantocellularis lateralis, and the hypothalamus. Evidence is presented that only the parabigeminal nucleus, the nucleus of the optic tract, and the posterior pretectal nucleus project to the superficial collicular layers (striatum griseum superficiale and stratum opticum), while all other afferents terminate in the deeper layers of the colliculus. Also presented is information concerning the rostrocaudal distribution of some of these afferent connections. These findings stress the multiplicity and diversity of inputs to the deeper collicular layers, and more specifically, identify multiple sources of the physiologically well-known representations of the somatic and auditory modalities in the colliculus.     

Comparative distribution of neuropeptide Y Y1 and Y5 receptors in the rat brain by using immunohistochemistry

Neuropeptide Y (NPY) Y1 and Y5 receptor subtypes mediate many of NPY's diverse actions in the central nervous system. The present studies use polyclonal antibodies directed against the Y1 and Y5 receptors to map and compare the relative distribution of these NPY receptor subtypes within the rat brain. Antibody specificity was assessed by using Western analysis, preadsorption of the antibody with peptide, and preimmune serum controls. Immunostaining for the Y1 and Y5 receptor subtypes was present throughout the rostral-caudal aspect of the brain with many regions expressing both subtypes: cerebral cortex, hippocampus, hypothalamus, thalamus, amygdala, and brainstem. Further studies using double-label immunocytochemistry indicate that Y1R immunoreactivity (-ir) and Y5R-ir are colocalized in the cerebral cortex and caudate putamen. Y1 receptor ir was evident in the central amygdala, whereas both Y1- and Y5-immunoreactive cells and fibers were present in the basolateral amygdala. Corresponding with the physiology of NPY in the hypothalamus, both Y1R- and Y5R-ir was present within the paraventricular (PVN), supraoptic, arcuate nuclei, and lateral hypothalamus. In the PVN, Y5R-ir and Y1R-ir were detected in cells and fibers of the parvo- and magnocellular divisions. Intense immunostaining for these receptors was observed within the locus coeruleus, A1-5 and C1-3 nuclei, subnuclei of the trigeminal nerve and nucleus tractus solitarius. These data provide a detailed and comparative mapping of Y1 and Y5 receptor subtypes within cell bodies and nerve fibers in the brain which, together with physiological and electrophysiological studies, provide a better understanding of NPY neural circuitries.     

Laminar structure of the dorsal lateral geniculate nucleus in the tree shrew (Tupaia glis)

Three tree shrews (Tupaia glis) were subjected to unilateral enucleation. Two of these were sacrificed seven months after surgery, and their brains were sectioned and stained for study of transneuronal atrophy within the dorsal lateral geniculate nucleus (LGN). The third animal was sacrificed ten days after enucleation; its brain was stained with a modified Nauta method for degenerating axons. Results of the two techniques are in good agreement. Ipsilateral and contralateral fibers were well segregated within the optic tracts. Transneuronal atrophy was found in layers 1 and 5 ipsilateral to the nucleated eye, and layers 2 and 4 contralaterally. Layer 3 did not appear to be differentially degenerated on the two sides, nor did a sixth layer, layer S, situated between layer 5 and the optic tract. Each superior colliculus receives fiber projections from both eyes, although those arising in the contralateral eye are far greater in number.     

Immunohistochemical localization of enkephalin in the central nervous system and pituitary of the lizard,Anolis carolinensis

The topographical distribution of enkephalin in the central nervous system of the lizard, Anolis carolinensis, has been studied by the immunoperoxidase technique with antiserum to leucine-enkephalin. Immunoreactive enkephalin perikarya, fibers and probably terminals are widely distributed throughout the central nervous system, which agrees well with the distribution of enkephalins in the mammalian brain. Enkephalin-containing perikarya are found in the subpallium (septum, nucleus accumbens, striatum, amydgala), preoptic and hypothalamic region, ventromedial nucleus and ventromedial area of thalamus, pretectal geniculate nucleus and posterodorsal nucleus of pretectum, nucleus of the lateral lemniscus, locus ceruleus, spinal trigeminal nucleus, nucleus of the solitary tract, medial parvocelluar nucleus, and dorsal horn of the spinal cord. Enkephalinergic fibers and terminals are found in the above–mentioned areas as well as in the pallium (medial and dorsal cortex, dorsal ventricular ridge), dorsomedial and anterior dorsolateral nucleus of the thalamus, habenua, nucleus of the stria medullaris, torus semicircularis, mesencephalic tegmental area, interpeducular nucleus, mesencephalic trigeminal nucleus, central gray, reticular formation, raphe nucleus, substantia nigra, isthmus region, and nucleus of the trapezoid body. Enkephalinergic pathways appear to exist between the septum and the medial cortex, nucleus accumbens and nucleus of the lateral olfactory tract, striatum and certain mesencephalic structures, hypothalamus and tegmentum, and between nucleus of the lateral lemniscus and torus semicirculais. In the pituitary, cells of the pars intermedia, and certain cells of the rostral pars distalis also show immunoreactivity to enkephalin antiserum. The distribution of enkephalin immunoreactivity throughout the hypothalamus and in the median eminence suggests involvement in neuroendocrine regulation. Presence of enkephalin in many extrahypothalamic brain areas indicates its important role in various sensory functions and in behavioral and autonomic integration.     

The projection to the mesencephalon from the dorsal column nuclei. An anatomical study in the cat

The terminal areas and cells of origin of the projection from the dorsal column nuclei to the mesencephalon were investigated by the intra-axonal transport method. Following injection of wheat germ agglutinin-horseradish peroxidase conjugate into the dorsal column nuclei, anterograde labeling was observed in several regions of the midbrain. The main terminal area was situated at the level of transition between the superior and inferior colliculus on the side contralateral to the injection site and comprised the intercollicular nucleus and part of the external and pericentral nuclei of the inferior colliculus and of the nucleus of the brachium of the inferior colliculus, but there were also projections to the caudal half of the deep and intermediate gray layers of the superior colliculus, the anterior and posterior pretectal nuclei, the nucleus of Darkschewitsch and nucleus ruber. Injections restricted to either the gracile nucleus or the cuneate nucleus revealed a somatotopic termination pattern in the intercollicular nucleus, superior colliculus and pretectal nuclei. The retrograde labeling seen after injection of tracer into the midbrain terminal areas showed that the cells of origin were located mainly in the rostral and caudal parts of the dorsal column nuclei, whereas the middle cell nest neurons were unlabeled, thus supporting previous observations that the neurons projecting to the midbrain constitute a population separate from that projecting to the thalamus. Cell counts revealed that the midbrain projection is of a considerable magnitude, involving between 10,000 and 15,000 neurons; its functional significance is, however, largely unknown.     

A comparison of dorsal column nuclei and spinal afferents in the European hedgehog (Erinaceus europeaus)

Lesions were made in the dorsal column nuclei of 19 hedgehogs, Erinaceus curopaeus. Lateral cervical hemisections (dorsal, lateral, and ventral funiculi on one side) were performed in an additional six. Nauta and Fink-Heimer staining techniques were utilized to follow fiber and terminal bouton degeneration. The dorsal column nuclei were found to have connections with the inferior olive, mesencephalic reticular formation, external nucleus of the inferior colliculus, medial pretectal nucleus, zona incerta, ventral nucleus of the lateral geniculate body, ventroposterior nucleus, and posterior complex of the thalamus. The spinal hemisections resulted in heavy degeneration in brain stem and mesencephalon but little in the thalamus. The impressively heavy termination of dorsal column fibers in PO, when contrasted with the meager projection of the rest of the cord, suggests that the VP-PO complex developed under the influence of the dorsal columns.     

Organization of subcortical pathways for sensory projections to the limbic cortex. II. Afferent projections to the thalamic lateral dorsal nucleus in the rat

Afferent projections to the thalamic lateral dorsal nucleus were examined in the rat by the use of retrograde axonal transport techniques. Small iontophoretic injections of horseradish peroxidase were placed at various locations within the lateral dorsal nucleus, and the location and morphology of cells of origin of afferent projections were identified by retrograde labeling. For all cases examined, subcortical retrogradely labeled neurons were most prominent in the pretectal complex, the intermediate layers of the superior colliculus, and the ventral lateral geniculate nucleus. Labeled cells were also seen in the thalamic reticular nucleus and the zona incerta. Within the cerebral cortex, labeled cells were prominent in the retrosplenial areas (areas 29b, 29c, and 29d) and the presubiculum. Labeled cells were also seen in areas 17 and 18 of occipital cortex. Peroxidase injections in the dorsal lateral part of the lateral dorsal nucleus result in labeled neurons in all of the ipsilateral pretectal nuclei, but especially those that receive direct retinal afferents. Labeled cells were also seen in the ventral lateral geniculate nucleus and the rostral tip of laminae IV-VI of the superior colliculus. In contrast, peroxidase injections in ventral medial portions of the lateral dorsal nucleus result in fewer labeled pretectal cells, and these labeled cells are found exclusively in the pretectal nuclei that do not receive retinal afferents. Other labeled cells following injections in the rostral and medial portions of the lateral dorsal nucleus are seen contralaterally in the medial pretectal region and nucleus of the posterior commissure, and bilaterally in the rostral tips of laminae IV and V of the superior colliculus. Camera lucida drawings of HRP labeled cells reveal that projecting cells in each pretectal nucleus have a characteristic soma size and dendritic branching pattern. These results are discussed with regard to the type of sensory information that may reach the lateral dorsal nucleus and then be relayed on to the medial limbic cortex.     

Aromatic L-amino acid decarboxylase-immunohistochemistry in the cat lower brainstem and midbrain

By indirect immunohistochemistry, the present study examined the distribution of neuronal structures in the cat medulla oblongata, pons, and midbrain, showing immunoreactivity to aromatic L-amino acid decarboxylase (AADC), which catalyzes the conversion of L-3, 4-dihydroxyphenylalanine (L-DOPA) to dopamine, and 5-hydroxytryptophan to serotonin (5HT). With simultaneous and serial double immunostaining techniques, immunoreactivity to this enzyme was demonstrated in most of the catecholaminergic and serotonergic neurons. We could also demonstrate AADC-IR cell bodies that do not contain tyrosine hydroxylase (TH-) or 5HT-immunoreactivity (called "D-type cells") outside such monoaminergic cell systems. At the medullo-spinal junction, very small D-type cells were found within and beneath the ependymal layer of the 10th area of Rexed surrounding the central canal. D-type cells were localized in the caudal reticular formation, nucleus of the solitary tract, a dorsal aspect of the lateral parabrachial nucleus, and pretectal areas as have been reported in the rat. Furthermore, the present study describes, in the cat brainstem, new additional D-type cell groups that have not been reported in the rat. Dense or loose clusters of D-type cells were localized in the external edge of the laminar trigeminal nucleus, dorsal motor nucleus of the vagus, external cuneate nucleus, nucleus praepositus hypoglossi, central, pontine, and periaqueductal gray, superficial layer of the superior colliculus, and area medial to the retroflexus. D-type cells were loosely clustered in the lateral part of the central tegmental field dorsal to the substantia nigra, extending dorsally in the medial division of the posterior complex of the thalamus and medial side of the brachium of the inferior colliculus. They extended farther rostrodorsally along the medial side of the nucleus limitans and joined with the pretectal cell group. Almost all these cells were very small and ovoid to round with 1-2 short processes with the exception of dorsal motor vagal cells. AADC-IR axons were clearly identified in the vagal efferent nerves, longitudinal medullary pathway, dorsal tegmental bundle rostral to the locus coeruleus. Serotonergic axons were identified not only in the central tegmentum field and lateral side of the central superior nucleus, but also in the ventral surface of the medulla oblongata. We describe principal densely stained fiber plexuses in the cat brainstem. The findings of the present study provide a morphological basis for neurons that decarboxylate endogenous and exogenous L-DOPA, 5HTP, and other aromatic L-amino acids.