Synaptic organization of septal projections in the rat medial habenula: a wheat germ agglutinin-horseradish peroxidase and immunohistochemical study.

The synaptic organization of septal inputs to the rat habenular complex of the dorsal diencephalon was examined employing the anterograde tracer wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). The cellular distribution of substance P (SP) and choline acetyltransferase (ChAT) immunoreactivity was also studied at the light and electron microscopic level. Following placements of tracer within the entire septum, labeled axons were observed in the stria medullaris and in the medial and lateral subnuclei of the habenula. Following injections of tracer in the nuclei triangularis and septofimbrialis of the posterior septum, the medial subnucleus was heavily labeled, whereas the lateral subnucleus was devoid of peroxidase activity. The medial subnucleus possessed labeled myelinated axons and terminals that contained clear, spherical vesicles and formed asymmetric contacts with dendritic spines and shafts. Terminals possessing WGA-HRP activity also formed non-synaptic junctions with other labeled or unlabeled terminals. SP and ChAT immunoreactivity in normal and colchicine-treated animals was confined to dendrites and somata within the medial habenula. Terminals containing clear spherical vesicles formed asymmetric synaptic contacts with these immunoreactive somatic and dendritic profiles. Based on the combined anterograde tracing and immunohistochemical data, it is proposed that septal projections provide a direct innervation to habenular neurons that contain ChAT or SP activity. These septal inputs may play an important role in the facilitation of the ChAT- and SP-positive habenular neurons, both of which provide prominent afferent inputs to the interpeduncular nucleus. Thus, neurons of the habenula and interpeduncular nucleus are under the direct and indirect influence of septal neurons within the limbic forebrain circuit.

Distribution and functional significance of Met-enkephalin-Arg6-Phe7- and Met-enkephalin-Arg6-Gly7-Leu8-like peptides in the blowfly Calliphora vomitoria. I. Immunocytochemical mapping of neuronal pathways in the brain.

Neuronal pathways immunoreactive to antisera against the extended-enkephalins, Met-enkephalin-Arg6-Phe7 (Met-7) and Met-enkephalin-Arg6-Gly7-Leu8 (Met-8), have been identified in the brain of the blowfly Calliphora vomitoria. Co-localisation with other enkephalins in certain neurons suggests that a precursor similar to preproenkephalin A exists in insects and that differential enzymatic processing occurs as in vertebrates. Co-localisations of the extended-enkephalin-like peptides with other vertebrate-type peptides, including cholecystokinin and pancreatic polypeptide, also occur. The enkephalinergic pathways are specific, comprising a few groups of highly characteristic neurons and areas of neuropil. Of special interest is the finding that parts of the antennal chemosensory and the optic lobe visual systems contain Met-8 immunoreactive neurons. Within the median neurosecretory cell groups, some of the giant neurons show immunoreactivity to Met-8 and others to both Met-8 and Met-7. Fibres from these cells project to the corpus cardiacum and also to the suboesophageal ganglion, where arborisations occur in the tritocerebral neuropil. Co-localisation studies of these cells have shown that at certain terminals, one particular type of peptide is the dominant neuroregulator, whilst at other terminals, within the same cell, a different co-synthesised peptide predominates. Several groups of lateral neurosecretory cells show clearly defined enkephalinergic pathways, most of which have connections with the central body. The complex patterns of immunoreactivity seen in terminals in the different parts of the central body, suggest an important role for the enkephalin-like peptides in the integration of multimodal sensory inputs. The physiological functions of the extended-enkephalin-like peptides in the brain of Calliphora is still unknown, but the anatomical evidence suggests they may have a role similar to that in mammals, where they are thought to control aspects of feeding behaviour.

Immunohistochemical characterization of pelvic neurons which project to the bladder, colon, or penis in rats.

Retrograde-tracing and immunohistochemical techniques were used in combination to investigate the types of putative transmitters in pelvic neurons that project to the bladder, colon or penis of rats. In addition, populations of axon varicosities associated with these neurons were characterized. Subpopulations of neurons in colchicine-treated major pelvic ganglia and accessory ganglia of male rats contained immunoreactivity (IR) for tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), or enkephalin (ENK), while types of immunoreactivity found in major groups of varicose axons were ENK, cholecystokinin (CCK), and somatostatin (SOM). Substance P (SP)-IR varicose axons were much less common. Bladder and colon neurons were similar in a number of ways. Many neurons contained NPY-IR (greater than or equal to 50%), fewer contained TH-IR (25-30%), and even fewer contained ENK-IR (5-15%) or VIP-IR (5-10%); many neurons were associated with baskets of ENK-IR varicosities (50-65%) and fewer neurons were surrounded by CCK- or SOM-IR varicosities (30-35%). Colon neurons differed from penis neurons in having a slightly larger proportion that contained ENK-IR (10-15%, compared with 1-3%). Penis neurons were markedly different from the other two groups in additional ways. More than 90% of them contained VIP-IR, whereas only 5-7% contained NPY-IR and none were immunoreactive for TH. Furthermore, although the proportion of penile neurons associated with many ENK-IR varicosities was similar to the bladder and colon neurons (45-50%), they were rarely seen close to CCK- or SOM-IR varicose axons. These studies describe similarities and differences in the histochemical properties of neurons which project to the bladder, colon, or penis and of the varicose axons associated with those neurons. This gives further insights into the possible transmitter mechanisms involved in the regulation of different pelvic functions.

Serotoninergic projections from the midbrain periaqueductal gray to the nucleus accumbens in the rat.

A combined method of retrograde tracing of horseradish peroxidase (HRP) and serotonin (5-HT) immunocytochemistry indicated that some 5-HT-like immunoreactive neurons in the midbrain periaqueductal gray (PAG) of the rat send their axons to the nucleus accumbens. These PAG neurons are mainly located in the ventromedial and ventrolateral subdivisions at the middle and caudal levels of PAG.

Origin of luteinizing hormone-releasing hormone neurons.

Neurons expressing luteinizing hormone-releasing hormone (LHRH), found in the septal-preoptic nuclei and hypothalamus, control the release of gonadotropic hormones from the anterior pituitary gland and facilitate reproductive behaviour. LHRH-expressing neurons are also found in the nervus terminalis, a cranial nerve that is a part of the accessory olfactory system and which projects directly from the nose to the septal-preoptic nuclei in the brain. During development, LHRH-immunoreactivity is detected in the peripheral parts of the nervus terminalis before it is found in the brain. Using a combination of LHRH immunocytochemistry and tritiated thymidine autoradiography in fetal mice, we show that LHRH neurons originate in the medial olfactory placode of the developing nose, migrate across the nasal septum and enter the forebrain with the nervus terminalis, arching into the septal-preoptic area and hypothalamus. Clinically, this migratory route for LHRH-expressing neurons could explain the deficiency of gonadotropins seen in 'Kallmann's syndrome' (hypogonadotropic hypogonadism with anosmia).

Brainstem origin of serotonin- and enkephalin-immunoreactive afferents to the opossum's cerebellum.

Previous studies have described the distribution of serotonin- and enkephalin-immunoreactive elements in the posterior lobe vermis of the opossum's cerebellum. In the present study we have used a double labeling paradigm which combines the retrograde transport of horseradish peroxidase (HRP) with serotonin and enkephalin immunohistochemistry to determine the brainstem origin of serotoninergic and enkephalinergic neurons that project to the opossum's cerebellar cortex. Subsequent to HRP injections into the posterior lobe vermis, widespread areas of the medulla and pons were found to contain retrogradely labeled neurons. Serotonin-immunoreactive somata are present primarily in the raphe nuclei and the adjacent reticular formation. Enkephalinergic neurons were numerous in the raphe nuclei, medial accessory olive, gigantocellular reticular formation, locus coeruleus, and the nucleus of the trapezoid body. However, serotoninergic neurons that project to the cerebellum were located only in the medullary pyramids and the reticular formation adjacent to the raphe. Double-labeled enkephalinergic neurons were located 1) within the medullary pyramids, 2) throughout the extent of the caudal medial accessory olive, 3) in the rostral subnucleus a of the medial accessory olive, 4) in the nucleus reticularis gigantocellularis pars ventralis, 5) in the nucleus reticularis lateralis, and 6) in the nucleus reticularis ventralis lateral to the inferior olivary complex. These results indicate that although neurons containing serotonin and enkephalin immunoreactivity may be present in some of the same pontine and medullary nuclei, those serotoninergic and enkephalinergic neurons that project to the cerebellum are present primarily in restricted and spatially separate regions of the caudal medulla.

Immunohistochemical demonstration of tyrosine hydroxylase (TH)-positive but dopamine beta-hydroxylase (DBH)-negative neuron-like cells in the pineal gland of golden hamsters.

Double immunostaining for tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) in the pineal gland revealed many TH-positive but DBH-negative, neuron-like cells in golden hamsters, but not in rats and gerbils. They were scattered in the meshwork of TH- and DBH-positive nerve fibers throughout the parenchyma. Their cell bodies were oval or spindle-shaped (maximum diameter: 15-20 micron). They had sometimes one or two short cell processes. Bilateral superior cervical ganglionectomy diminished strikingly the number of TH- and DBH-positive nerve fibers, but did not affect the number of TH-positive cells.

Central serotonergic projections to the nucleus tractus solitarii: evidence from a double labeling study in the rat.

Projections from several brainstem serotonergic nuclei to the nucleus tractus solitarii were investigated in the rat. Experiments were performed using a double labeling method combining retrograde radioautographic tracing and serotonin immunohistochemistry. After injection of the radioactive tracer ([3H] wheat germ agglutinin) into the lateral nucleus tractus solitarii, nerve cell bodies exhibiting both radioautographic labeling and immunostaining were detected in all the serotonergic nuclei investigated, namely the nucleus raphe magnus, the ventromedial paragigantocellular nucleus, the nuclei raphe pontis, medianus and dorsalis, the medial lemniscus and the reticulotegmental nucleus of the pons. Most of the double labeled perikarya observed were in the nucleus raphe magnus, the adjacent part of the paragigantocellular nucleus and the nucleus raphe dorsalis. Nerve cell bodies retrogradely labeled but devoid of immunostaining were also observed, together with the double labeled perikarya, within serotonergic nuclei. These results provide direct evidence that brainstem serotonergic neurons contribute to the innervation of the nucleus tractus solitarii. They indicate that the nucleus raphe magnus and the nucleus raphe dorsalis constitute two major sources of central serotonergic projections to the nucleus tractus solitarii.

Evidence for a projection from the B9 serotonergic cell group to the median raphe nucleus.

Following injections of several fluorescent retrograde tracers into the median raphe nucleus large numbers of retrogradely labeled cells were observed in the ventral mesencephalic tegmentum. Subsequent immunocytochemical processing for serotonin-like immunoreactivity suggested that a large component of this projection originates from serotonergic B9 cells. Although tracer injections into the dorsal raphe did not result in similar labeling of B9 cells, evidence was obtained suggesting the existence of a small serotonergic projection from the median to the dorsal raphe.

Immunocytochemical study of the hypothalamic magnocellular neurosecretory nuclei of the snake Natrix maura and the turtle Mauremys caspica.

An immunocytochemical study of the magnocellular neurosecretory nuclei was performed in the snake Natrix maura and the turtle Mauremys caspica by use of antisera against: (1) a mixture of both bovine neurophysins, (2) bovine oxytocin-neurophysin, (3) arginine vasotocin, and (4) mesotocin. Arginine vasotocin- and mesotocin-immunoreactivities were localized in individual neurons of the supraoptic and paraventricular nuclei, with a distinct pattern of distribution in both species. The same cells appeared to be stained by the anti-oxytocin-neurophysin and antimesotocin sera. The supraoptic nucleus can be subdivided into rostral medial and caudal portions. In N. maura, but not in M. caspica, neurophysin-immunoreactive neurons were found in the retrochiasmatic nucleus. No immunoreactive elements were seen in the suprachiasmatic nucleus of both species after the use of any of the antisera. A dorsolateral aggregation of neurophysin-containing cells, localized over the lateral forebrain bundle, was present in both species. Magnocellular and parvocellular neurophysin-immunoreactive neurons were present in the paraventricular nucleus of both species. In the turtle, the paraventricular neurons were arranged into four distinct layers parallel to the ependyma; these neurons were bipolar with the major axis perpendicular to the ventricle, and many of them projected processes toward the cerebrospinal-fluid compartment. In N. maura a group of large neurons of the paraventricular nucleus was found in a very lateral position. The posterior lobe of the hypophysis and the external zone of the median eminence contained arginine vasotocin- and mesotocin-immunoreactive nerve fibers. The lamina termialis of both species was supplied with a dense bundle of fibers containing immunoreactive neurophysin. Neurophysin-immunoreactive fibers were also present in the septum, some telencephalic regions, including the cortex and the olfactory tubercule, in the paraventricular organ, and the periventricular and periaqueductal gray of the brainstem.

Noradrenergic neurons with divergent projections to the motor trigeminal nucleus and the spinal cord: a double retrograde neuronal labeling study.

Double retrograde axonal tracing was combined with the indirect immunofluorescence antibody method to determine whether noradrenergic neurons have divergent projections to the motor nucleus of the trigeminal nerve and the spinal cord. Rhodamine-labeled microspheres were injected into the motor trigeminal nucleus and True Blue was deposited into lumbar segments of the spinal cord. After a 10-18-day survival period, brainstem sections were processed for immunofluorescence staining of noradrenergic neurons using antibodies to rat dopamine-beta-hydroxylase. Rhodamine-labeled noradrenergic neurons were observed ipsilaterally throughout the A5 and A7 groups; the contralateral A5 and A7 groups contained few rhodamine-labeled cells. A few rhodamine-labeled noradrenergic neurons were observed in the locus coeruleus and subcoeruleus. True Blue-labeled noradrenergic neurons were identified in the A5 and A7 groups, in the ventral part of the locus coeruleus and in the subcoeruleus. Double retrogradely labeled noradrenergic neurons were observed in the A5 and A7 groups but not in the locus coeruleus and subcoeruleus. Of the total number of rhodamine-labeled noradrenergic cells, a large percentage also contained True Blue: 54% in the caudal A5 group, 59% in the rostral A5 group, and 72% in the A7 group. Of the total number of True Blue-labeled noradrenergic neurons, the percentage of double retrogradely labeled cells was 33% in the caudal A5 group, 46% in the rostral A5 group, and 56% in the A7 group. The findings of this study provide the first anatomic evidence for the existence of a prominent population of noradrenergic cells in the A5 and A7 groups with divergent projections to the motor trigeminal nucleus and the spinal cord. We propose that this subpopulation of noradrenergic neurons in the A5 and A7 groups influences motoneurons at multiple levels of the neuraxis.

A direct demonstration of the perforant pathway terminal zone in Alzheimer's disease using the monoclonal antibody Alz-50.

The perforant pathway links the entorhinal cortex with the hippocampal formation and provides this structure with its major cortical input. The cells of origin of the perforant pathway are destroyed in Alzheimer's disease (AD) and a marked depletion of glutamate, the putative neurotransmitter in its terminal zone, occurs. We report that the monoclonal antibody Alz-50 recognizes an antigen in the terminal zone of the perforant pathway in AD. This observation provides direct evidence for the involvement of the perforant pathway in AD, and demonstrates that Alz-50 can be used to study neural connectivity in AD brains.

Origin, distribution and organization of the serotoninergic innervation in the inferior olivary complex of the rat.

The serotoninergic innervation of the inferior olivary complex of the rat was studied using a specific immunohistochemical technique. Fibers and varicosities positive for serotonin were present throughout the nucleus. The densest varicosities were found in the lateral portion of the dorsal accessory olive and the caudal portion of the medial accessory olive. The rostral medial accessory olive and the principal olive were sparsely populated with labeled elements. Ultrastructurally, labeled profiles were found in close opposition to small dendrites and to olivary cell bodies, but they did not display any synaptic specialization. Labeled perikaria were found in the periolivary regions, some of them located laterally to the olivary complex are responsible for the serotoninergic innervation of the dorsal accessory olive; some others located dorsally and medially in the nucleus raphe obscurus and raphe pallidus were responsible for the innervation of the medial accessory olive.

Distribution and origin of vasoactive intestinal polypeptide (VIP)-immunoreactive, acetylcholinesterase-positive and adrenergic nerves of the cerebral arteries in the bent-winged bat (Mammalia: Chiroptera).

The overall distribution and origins of vasoactive intestinal polypeptide (VIP)-immunoreactive (IR), acetylcholinesterase (AChE)-positive and adrenergic nerves in the walls of the cerebral arteries were investigated in the bent-winged bat. VIP-IR and AChE-positive nerves innervating the bat cerebral vasculature appear to arise mainly from VIP-IR and AChE-positive cell bodies within microganglia found in the nerve bundle accompanying the sympathetic nerve bundle within the tympanic cavity. These microganglia, as well as the nerve bundle containing them, do not emit catecholamine fluorescence, suggesting that they are of the cranial parasympathetic outflow, probably the facial or glossopharyngeal one. The axons from VIP-IR and AChE-positive microganglia run intermingled with sympathetic adrenergic nerves in the same thick fiber bundles, and reach the cranial cavity through the carotid canal. In addition, some of the VIP-IR fibers innervating the vertebro-basilar system, at least the basilar artery, originate from VIP-IR nerve cells located in the wall of this artery. The supply of VIP-IR fibers to the bat major cerebral arteries is the richest among mammals in that it is much greater in the vertebro-basilar system than in the internal carotid system: plexuses of VIP-IR nerves are particularly dense along the walls from the posterior ramus to posterior cerebral and basilar arteries. Small pial and intracerebral arteries of the vertebro-basilar system, especially those of the posterior cerebral artery which supply most parts of the diencephalon and cerebrum, are also richly innervated by peripheral VIP-IR fibers. This pattern corresponds well with the innervation pattern of adrenergic and AChE-positive nerves.

Projection of neurotensin-like immunoreactive neurons from the lateral parabrachial area to the central amygdaloid nucleus of the rat with reference to the coexistence with calcitonin gene-related peptide.

The origin of neurotensin-like immunoreactive (NTI) fibers in the central amygdaloid nucleus (AC) in the rat was examined using indirect immunofluorescence and retrograde tracing combined with immunocytochemistry. Destruction of the external subdivision of the lateral parabrachial nucleus, which contains a group of NTI neurons, resulted in a marked reduction of these fibers in the ipsilateral AC, which suggests that most of these fibers are of extrinsic origin. This was also supported by the finding that injection of fast blue dye into the AC labeled many neurons in the external subdivision of the lateral parabrachial nucleus ipsilaterally, and that simultaneous treatment with antiserum against NT stained some of these neurons. Subsequent immunohistochemical staining of alternate sections revealed that many of these NTI neurons were also labeled by calcitonin gene-related peptide antiserum.

Neuronal pathways to the rat thyroid revealed by retrograde tracing and immunocytochemistry.

The distribution and origin of the nerve fibres innervating the rat thyroid were studied by immunocytochemistry, retrograde tracing and denervation experiments. Immunocytochemistry revealed nerve fibres containing noradrenaline, neuropeptide Y, vasoactive intestinal peptide, peptide histidine-isoleucine, galanin, substance P, neurokinin A and calcitonin gene-related peptide around blood vessels and follicles. Many of these transmitter candidates were found to co-exist with each other in different combinations in different subpopulations of neurons. Sympathectomy eliminated all noradrenaline- and noradrenaline/neuropeptide Y-containing fibres in the thyroid. Cervical vagotomy eliminated about 50% of the galanin-, substance P- and calcitonin gene-related peptide-containing fibres. Local denervation (removal of the thyroid ganglion and the thyroid nerve) eliminated all galanin- and substance P-immunoreactive fibres and the majority of noradrenaline-, noradrenaline/neuropeptide Y-, vasoactive intestinal peptide- and calcitonin gene-related peptide-containing fibres in the thyroid gland. Injection of True Blue into the thyroid gland labelled cell bodies in the thyroid ganglion, the laryngeal ganglion, the superior cervical ganglion, the jugular-nodose ganglionic complex, the dorsal root ganglia (C2-C5) and the trigeminal ganglion. Judging from the number of labelled nerve cell bodies, the superior cervical ganglion and the thyroid ganglion contribute most to the thyroid innervation, while the laryngeal ganglion and the trigeminal ganglion contribute least. The True Blue-labelled ganglia were examined for the presence of various populations of nerve cell bodies (only major populations are listed). The thyroid ganglion harboured neuropeptide Y, vasoactive intestinal peptide and galanin/vasoactive intestinal peptide cell bodies (in order of predominance); the laryngeal ganglion galanin/vasoactive intestinal peptide, vasoactive intestinal peptide and calcitonin gene-related peptide cell bodies; the superior cervical ganglion noradrenaline/neuropeptide Y and noradrenaline cell bodies; the jugular ganglion calcitonin gene-related peptide, substance P/calcitonin gene-related peptide and galanin/substance P/calcitonin gene-related peptide cell bodies; the nodose ganglion vasoactive intestinal peptide and vasoactive intestinal peptide/galanin cell bodies; the dorsal root ganglia (C2-C5) and the trigeminal ganglion calcitonin gene-related peptide, substance P/calcitonin gene-related peptide and galanin/substance P/calcitonin gene-related peptide cell bodies.(ABSTRACT TRUNCATED AT 400 WORDS)

The neostriatal mosaic: III. Biochemical and developmental dissociation of patch-matrix mesostriatal systems.

In the previous paper (Gerfen et al., 1987) mesostriatal dopaminergic neurons were shown to be subdivided into dorsal and ventral tiers that project to the striatal matrix and patch compartments, respectively. The present study provides experimental evidence that these patch-matrix mesostriatal dopaminergic systems are biochemically and developmentally distinct. A 28 kDa calcium-binding protein (CaBP, or calbindin-D28 kDa) is expressed in dorsal tier mesostriatal dopaminergic neurons. The distribution of such neurons, located in the ventral tegmental area, dorsal tier of the substantia nigra pars compacta, and retrorubral area, matches that of dopaminergic neurons that project to the striatal matrix. Dopaminergic neurons that do not express CaBP--those in the ventral tier of the pars compacta and in the pars reticulata--are distributed in a pattern that matches the origin of the dopaminergic projection to the striatal patches. During development, dopaminergic afferents to the striatal patch compartment are in place prior to the development of those to the matrix. Injections of the neurotoxin 6-hydroxydopamine (6-OHDA) into the striatum of newborn rats result in a selective and long-lasting depletion of dopaminergic afferents in the striatal patches. The later-developing matrix projection is relatively spared by such lesions. The distribution of surviving dopaminergic neurons, labeled with tyrosine hydroxylase (TH) immunoreactivity, matches the pattern of dorsal tier neurons previously shown to provide inputs to the matrix. Surviving neurons also express CaBP immunoreactivity and have dendrites that spread mediolaterally, in the plane of the pars compacta. On the other hand, those neurons that project to the patches are selectively lesioned by the neonatal 6-OHDA striatal injections, do not express CaBP, and have dendrites that are directed ventrally into the pars reticulata.

The dentato-olivary projection in the rat as a presumptive GABAergic link in the olivo-cerebello-olivary loop. An ultrastructural study.

It is here shown that autoradiographically labelled axon terminals of the dentato-olivary projection form a heterogeneous population. However, a majority of them constitute an even class of synapses, characterized by their small axonal size, their content in pleimorphic vesicles, and the establishment of symmetric synapses on small dendrites, about 5% of which are linked through a gap junction. The same material, used for immunocytochemistry of GABA with the postembedding technique, discloses that a majority of boutons with cytological features similar to the dentato-olivary terminals are GABA-immunoreactive, especially those synapsing on dendrites linked by gap junctions. The cerebello-olivary projection, despite its heterogeneity, thus appears as part of the GABAergic system which governs the synaptic modulation of the electrotonic coupling between olivary neurons.

Vestibulospinal pathway in rabbit includes GABA-synthesizing neurons.

When Fast blue is injected into the rabbit spinal cord it is retrogradely transported into nerve cell bodies located in the medial and the descending vestibular nuclei. Approximately 50% of the Fast blue-positive cells also contain glutamic acid decarboxylase-like immunoreactivity. These neurons presumably synthesize gamma-aminobutyric acid (GABA) and the rabbit vestibulospinal pathway therefore contains a substantial inhibitory component.