Ultrastructural single- and double-label immunohistochemical studies of substance P-containing terminals and dopaminergic neurons in the substantia nigra in pigeons.

The vast majority of striatonigral projection neurons in pigeons contain substance P (SP), and the vast majority of SP-containing fibers terminating in the substantia nigra arise from neurons in the striatum. To help clarify the role of striatonigral projection neurons, we conducted electron microscopic single- and double-label immunohistochemical studies of SP+ terminals and/or dopaminergic neurons (labeled with either anti-dopamine, DA, or anti-tyrosine hydroxylase, TH) in pigeons to determine: (1) the synaptic organization of SP+ terminals, (2) the synaptic organization of TH+ perikarya and/or dendrites, and (3) the synaptic relationship between SP+ terminals and TH+ neurons in the substantia nigra. Tissue single-labeled for SP revealed numerous SP+ terminals contacting thin unlabeled dendrites in the substantia nigra, but few SP+ terminals were observed contacting perikarya or large-diameter dendrites. SP+ terminals contained round, densely packed, clear vesicles, and often contained one or more dense-core vesicles. Synaptic junctions between SP+ terminals and their targets were more often symmetric (86%) than asymmetric. In tissue single-labeled for DA, we observed few terminals contacting DA+ perikarya, whereas terminals contacting DA+ dendrites were more abundant. Terminals contacting DA+ structures comprised at least four different morphologically distinct types based on the morphology of the clear synaptic vesicles and the type of synaptic junction. One type of terminal contained round clear vesicles and made symmetric synapses, and thus resembled the predominant type of SP+ terminal. The second type contained round clear vesicles and made asymmetric synapses, the third type contained medium-size pleomorphic clear vesicles and made symmetric synapses, and the fourth type contained small pleomorphic clear vesicles and made symmetric synapses. The presence of contacts between SP+ terminals and dopaminergic dendrites in the substantia nigra was directly demonstrated in tissue double-labeled for SP (by the peroxidase-antiperoxidase procedure, or PAP, with diaminobenzidine) and TH (by either the silver-intensified immunogold procedure or the PAP procedure with benzidine dihydrochloride). SP+ terminals commonly contacted thin TH+ dendrites in the substantia nigra, but few SP+ terminals contacted large-diameter TH+ dendrites or perikarya. Synapses between SP+ terminals and TH+ neurons were always symmetric. TH+ dendrites also were contacted by terminals not labeled for SP, which were more abundant than were SP+ terminals. Non-TH+ neurons were also contacted by both SP+ terminals and non-SP+ terminals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructural evidence of dopaminergic input to enkephalinergic neurons in rat neostriatum

The synaptic relationship between neuronal structures reacting with antibodies to tyrosine hydroxylase (TH) and Leu- or Met-enkephalin (ENK) was studied by the 'mirror technique' in adjacent sections of rat neostriatum. TH-immunoreactive (TH-IR) axonal boutons surrounding the neural perikarya and proximal dendrites of ENK-immunoreactive (ENK-IR) neurons were very thin (0.1-0.4 micrograms). They contained many small clear vesicles and sometimes had symmetrical membrane specializations. This provides morphological evidence for catecholaminergic, presumably dopaminergic inputs to rat striatal enkephalin neurons.     

Innervation of substance P neurons by catecholaminergic terminals in the neostriatum

In this study we tried to establish by the electron microscopic 'mirror technique' whether substance P-like immunoreactive neurons in the neostriatum receive synaptic inputs from catecholaminergic, presumably dopaminergic, nigrostriatal axons. Tyrosine hydroxylase-immunoreactive (TH-IR) axonal boutons were in synaptic contact with the somas and proximal dendrites of SP-IR neostriatal neurons, which are medium in size and have unindented nuclei. This suggests that nigrostriatal dopaminergic neurons monosynaptically influence the strionigral substance P neurons.     

Ultrastructural characterization of the mesostriatal dopamine innervation in mice, including two mouse lines of conditional VGLUT2 knockout in dopamine neurons

Despite the increasing use of genetically modified mice to investigate the dopamine (DA) system, little is known about the ultrastructural features of the striatal DA innervation in the mouse. This issue is particularly relevant in view of recent evidence for expression of the vesicular glutamate transporter 2 (VGLUT2) by a subset of mesencephalic DA neurons in mouse as well as rat. We used immuno-electron microscopy to characterize tyrosine hydroxylase (TH)-labeled terminals in the core and shell of nucleus accumbens and the neostriatum of two mouse lines in which the Vglut2 gene was selectively disrupted in DA neurons (cKO), their control littermates, and C57BL/6/J wild-type mice, aged P15 or adult. The three regions were also examined in cKO mice and their controls of both ages after dual TH-VGLUT2 immunolabeling. Irrespective of the region, age and genotype, the TH-immunoreactive varicosities appeared similar in size, vesicular content, percentage with mitochondria, and exceedingly low frequency of synaptic membrane specialization. No dually labeled axon terminals were found at either age in control or in cKO mice. Unless TH and VGLUT2 are segregated in different axon terminals of the same neurons, these results favor the view that the glutamatergic cophenotype of mesencephalic DA neurons is more important during the early development of these neurons than for the establishment of their scarce synaptic connectivity. They also suggest that, in mouse even more than rat, the mesostriatal DA system operates mainly through non-targeted release of DA, diffuse transmission and the maintenance of an ambient DA level.     

Dopaminergic terminals form synaptic contacts with enkephalinergic striatal neurons in pigeons: an electron microscopic study

Medium spiny projection neurons of the striatum consist of two major neuropeptide-specific types, one type containing substance P and another type containing enkephalin. Both of these types have been shown to receive dopaminergic input onto their perikarya and proximal dendrites. However, whether each of these types receives direct dopaminergic input onto distal dendritic shafts and onto dendritic spines has not been explored in depth. In the present study, we used electron microscopic immunohistochemical double-label techniques to examine the synaptic organization of dopaminergic input onto enkephalin-positive (ENK +) striatal neurons in pigeons, in whom ENK + striatal perikarya, dendritic shafts and spines can be readily labeled. Antibodies against tyrosine hydroxylase were used to label dopaminergic terminals using a silver-intensified immunogold method. ENK + neurons were labeled using diaminobenzidine. We found that dopaminergic terminals make appositions and form symmetric synapses with the perikarya, dendritic shafts, and dendritic spine necks of ENK + striatal neurons. Thus, nigral dopaminergic neurons provide a monosynaptic input onto ENK + striatal neurons in a manner similar to that described previously by us for substance P-positive striatal medium spiny neurons.     

Cholinergic innervation of olfactory glomeruli in the rat: An ultrastructural immunocytochemical study

The ultrastructural organization of cholinergic afferents to the rat olfactory bulb (OBI) was studied with the aid of choline acetyltransferase (ChAT) immunocytochemistry in electron microscopy. Particular attention has been paid to a subset of glomeruli characterized by a remarkably high density of cholinergic afferents. Numerous cholinergic terminals making symmetric or asymmetric synaptic contacts were observed in the periglomerular area. ChAT-labelled terminals have a diameter ranging from 0.3 to 1.5 μm and contain numerous clear agranular vesicles. Axo-somatic and axo-dendritic contacts were both observed in contact with several types of target neurons. Three types of cholinoceptive, noncholinergic neurons could be identified: periglomerular cells, superficial short-axon cells, and external tufted cells. Our results provide an anatomical substrate for the hypotheses concerning the complex effects of acetylcholine in the processing of sensory information in the olfactory bulb. © 1993 Wiley-Liss, Inc.     

The immunocytochemical localization of substance P in the human striatum: a postmortem ultrastructural study

The striatum is a basal ganglia structure that is involved in motor, cognitive, and behavioral functions. In the striatum, the neuroactive peptide, substance P, is colocalized with GABA in the subset of medium spiny neurons that projects to the substantia nigra. Normal human striata (n = 5) obtained from the Maryland Brain Collection were processed for substance P immunoreactivity, prepared for electron microscopy, and analyzed using both stereology and simple profile counts. Most substance P-labeled neurons had a nonindented nucleus and a moderate amount of cytoplasm, typical of medium spiny projection neurons in other species. A small percentage (8%) of labeled neurons had indented nuclei, but otherwise had similar morphology. Synapses formed on labeled cell bodies were rare. Synapses formed by substance P-labeled axon terminals constituted 4.4% of the total synapses in the neuropil. Labeled terminals (1) formed synapses with both spines and dendrites with approximately equal frequency, (2) formed mostly symmetric synapses (76-85%), and (3) formed synapses predominantly with unlabeled (78%) profiles. Substance P-labeled spines varied in shape and comprised 37-42% of all spines forming synapses. In the caudate, the proportion of synapses with perforated postsynaptic densities was 55% on unlabeled vs. 45% on labeled spines, but in the putamen, this type of synapse was much more frequently present on unlabeled (73%) vs. labeled (27%) spines. These data describe substance P in the normal human striatum, which serve as comparative data to that of other species as well as normative data for further studies of brain disease that may involve striatal substance P neurons.     

Development of substance P-containing neurons in the central nervous system in mice: an immunocytochemical study

The embryonic development of substance P (SP) in the central nervous system (CNS) of mice has been studied with the use of peroxidase antiperoxidase (PAP) immunocytochemistry. Immature SP-positive cells initially appear at embryonic day 12 (E12) in the epithalamus and in a column of cells extending from the myelencephalon throughout the length of the neural tube. By E13, SP-positive cells appear in the amygdaloid nuclear complex, the bed nucleus of the stria terminalis, and in the caudal medulla. Fibers are first detected in the stria terminalis at this age. Over the next 48 hours, a plethora of SP-positive cells appears throughout the CNS, notably in the septal area, diagonal band nucleus, piriform cortex, accumbens nucleus, hypothalamus, rostral striatum, superior and inferior colliculi, intercollicular nucleus, substantia nigra, interpeduncular nucleus, vestibular nuclei, spinal nucleus of the trigeminal, and the nucleus of the tractus solitarii. Subsequently, SP-positive neurons and fibers increase in number and staining intensity except in the medullary raphe where the apparent number of SP-positive neurons decreases after E16. Whereas the pattern of SP staining is quite similar in mice and rats, the time of initial detection of SP-like immunoreactivity in specific nuclei is 1-4 days earlier in mice than that reported in rats with different antisera.     

Cholinergic innervation of the rat dentate gyrus: an immunocytochemical and electron microscopical study

Immunocytochemical studies using a monoclonal antibody to choline acetyltransferase (ChAT) were performed on sections of rat dentate gyrus. Light microscopical analysis of the immunoreactivity revealed dense fiber networks and many punctate structures predominantly located at the interface of the granule cell layer and molecular layer. In the elctron microscope, the immunostained punctate structures were identified as synaptic boutons which formed mainly symmetrical contacts onto dendritic elements. Few ChAT-immunoreactive boutons formed axosomatic contacts.     

Cholinergic neurons in the rat septal complex: ultrastructural characterization and synaptic relations with catecholaminergic terminals.

Physiological and pharmacological studies have suggested that catecholamines modulate cholinergic neurons in the medial septal and diagonal band nuclei (i.e., the septal complex). Thus, the ultrastructural morphology of neurons containing choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine, and their relation to catecholaminergic terminals exhibiting immunoreactivity for the catecholamine synthesizing enzyme tyrosine hydroxylase (TH) were examined in the rat septal complex. Dual immunoautoradiographic and peroxidase anti-peroxidase labeling methods were used to simultaneously localize antibodies raised in rabbits against TH and from rat-mouse hybridomas against ChAT in single sections. At least two types of perikarya with ChAT-immunoreactivity (ChAT-I) were observed. The first type were large (20-30 microns), elongated or round, and contained a small indented nucleus with an abundant cytoplasm and an occasional lamellar body. The second type was also either ovoid or round but was medium-sized (15-20 microns) and contained a larger indented nucleus and a smaller amount of cytoplasm than the first type. Both types of perikarya as well as dendrites with ChAT-I were surrounded by astrocytic processes apposed to most of their plasmalemmal surfaces. The distribution and types of terminal associations (i.e., asymmetric synapses, symmetric synapses and appositions which lacked a membrane specialization in the plane of section analyzed) with ChAT-labeled perikarya and dendrites were quantitatively evaluated. The majority (68% of 197) of the presynaptic terminals were unlabeled; the remaining terminals were immunoreactive for TH (25%) or ChAT (7%). All three types of terminals contacted primarily the shafts of small dendrites and more rarely ChAT-labeled perikarya and large dendrites. ChAT-labeled terminals: (1) formed associations with unlabeled perikarya and dendrites (31% of 176); (2) formed associations with perikarya and dendrites with ChAT-I (7%); (3) contacted the same unlabeled perikarya and dendrite as a TH-containing terminal (21%); (4) were in apposition to TH-labeled terminals (25%); or (5) were either in apposition to unlabeled or ChAT-labeled terminals or lacked associations with any processes. The majority of associations formed by the terminals with ChAT-I were on the shafts of small dendrites. Moreover, most of the associations formed were either symmetric synapses or appositions not separated by astrocytes in the plane of section analyzed. These findings provide cellular substrates in the septal complex (1) for sparse synaptic input relative to astrocytic investment of cholinergic neurons and (2) for direct synaptic modulation of cholinergic and non-cholinergic neurons by catecholamines and/or acetylcholine. These findings have direct relevance to catecholaminergic-cholinergic interactions and to the neuropathological basis for Alzheimer's disease.     

Columnar distribution of catecholaminergic neurons in the ventrolateral periaqueductal gray and their relationship to efferent pathways

The periaqueductal gray (PAG) is a critical brain region involved in opioid analgesia and provides efferents to descending pathways that modulate nociception. In addition, the PAG contains ascending pathways to regions involved in the regulation of reward, including the substantia nigra (SN) and the ventral tegmental area (VTA). SN and VTA contain dopaminergic neurons that are critical for the maintenance of positive reinforcement. Interestingly, the PAG is also reported to contain a population of dopaminergic neurons. In this study, the distribution of catecholaminergic neurons within the ventrolateral (vl) PAG was examined using immunocytochemical methods. In addition, the catecholaminergic PAG neurons were examined to determine whether these neurons are integrated into ascending (VTA, SN) and descending rostral ventral medulla (RVM) efferent pathways from this region. The immunocytochemical analysis determined that catecholaminergic neurons in the PAG are both dopaminergic and noradrenergic and these neurons have a distinct rostrocaudal distribution within the ventrolateral column of PAG. Dopaminergic neurons were concentrated rostrally and were significantly smaller than noradrenergic neurons. Combined immunocytochemistry and tract tracing methods revealed that catecholaminergic neurons are distinct from, but closely associated with, both ascending and descending efferent projection neurons. Finally, by electron microscopy, catecholaminergic neurons showed close dendritic appositions with other neurons in PAG, suggesting a possible nonsynaptic mechanism for regulation of PAG output by these neurons. In conclusion, our data indicate that there are two populations of catecholaminergic neurons in the vlPAG that form dendritic associations with both ascending and descending efferents suggesting a possible nonsynaptic modulation of vlPAG neurons. Synapse, 2013. © 2012 Wiley Periodicals, Inc.     

Coexistence of oxytocin and tyrosine hydroxylase in the rat hypothalamus,an immunocytochemical study

Summary Immunocytochemical double labelling was used to determine the structural relationship of oxytocin (OT) and tyrosine hydroxylase (TH) containing perikarya and processes in the rat hypothalamus. Extrahypothalamic TH fibers, as well as parvocellular TH neurons were found to form contacts with OT cells. A fraction of the OT neurons contained TH immunoreactivity. It is likely that in addition to the classical mesencephalic afferences also hypothalamic interneurons and magnocellular dopaminergic neurons control the hypothalamo neurohypophysial system.     

Functional fetal nigral grafts in a patient with Parkinson's disease: Chemoanatomic,ultrastructural, and metabolic studies

A patient with Parkinson's disease received bilateral fetal human nigral implants from six donors aged 6.5 to 9 weeks post-conception. Eighteen months following a post-operative clinical course characterized by marked improvement in clinical function, this patient died from events unrelated to the grafting procedure. Post-mortem histological analyses revealed the presence of viable grafts in all 12 implant sites, each containing a heterogeneous population of neurons and glia. Approximately 210,146 implanted tyrosine hydroxylase-immunoreactive (TH-ir) neurons were found. A greater number of TH-ir grafted neurons were observed in the right (128,162) than the left (81,905) putamen. Grafted TH-ir neurons were organized in an organotypic fashion. These cells provided extensive TH-ir and dopamine transporter-ir innervation to the host striatum which occurred in a patch-matrix fashion. Quantitative evaluations revealed that fetal nigral grafts reinnervated 53% and 28% of the post-commissural putamen on the right and left side, respectively. Grafts on the left side innervated a lesser area of the striatum, but optical density measurements were similar on both sides. There was no evidence that the implants induced sprouting of host TH-ir systems. Electron microscopic analyses revealed axo-dendritic and occasional axo-axonic synapses between graft and host. In contrast, axo-somatic synapses were not observed. In situ hybridization for TH mRNA revealed intensely hybridized grafted neurons which far exceeded TH mRNA expression within residual host nigral cells. In addition, γ-amino butyric acid (GABA)-ergic neurons were observed within the graft that formed a dense local neuropil which was confined to the implant site. Serotonergic neurons were not observed within the graft. Cytochrome oxidase activity was increased bilaterally within the grafted post-commissural putamen, suggesting increased metabolic activity. In this regard, a doubling of cytochrome oxidase activity was observed within the grafted post-commissural putamen bilaterally relative to the non-grafted anterior putamen. The grafts were hypovascular relative to the surrounding striatum and host substantia nigra. Blood vessels within the graft stained intensely for GLUT-1, suggesting that this marker of blood-brain barrier function is present within human nigral allografts. Taken together, these data indicate that fetal nigral neurons can survive transplantation, functionally reinnervate the host putamen, establish synaptic contacts with host neurons, and sustain many of the morphological and functional characteristics of normal nigral neurons following grafting into a patient with PD. © 1996 Wiley-Liss, Inc.     

Neurotensin terminals form synapses primarily with neurons lacking detectable tyrosine hydroxylase immunoreactivity in the rat substantia nigra and ventral tegmental area.

A light and electron microscopic double antigen localization technique was employed to examine the fine structural relationship between neurotensin-containing axon terminals and dopaminergic neurons in the substantia nigra and ventral tegmental area of the rat. At the light microscopic level, neurotensin-immunoreactive terminals were densely distributed throughout the substantia nigra pars compacta and ventral tegmental area in close proximity to tyrosine hydroxylase-immunoreactive somata and dendrites. On electron microscopic examination, direct synaptic connections were identified between neurotensin-immunoreactive axon terminals and tyrosine hydroxylase-immunopositive perikarya and dendrites. However, only 8.2% and 8.8% of the neurotensin-immunoreactive axonal profiles detected in the substantia nigra and ventral tegmental area, respectively, were found in direct apposition with tyrosine hydroxylase-immunostained elements. In turn, only 9.3% and 10.0% of tyrosine hydroxylase immunoreactive dendrites sampled from the substantia nigra and ventral tegmental area, respectively, were seen in contact with neurotensin immunopositive axon terminals. However, neurotensin-immunoreactive and tyrosine hydroxylase-immunolabelled elements were frequently identified in close anatomical proximity (less than 5 microns) to one another. These results are interpreted in light of the selective association of neurotensin receptors with dopaminergic neurons in the substantia nigra and ventral tegmental area to suggest a predominantly parasynaptic mechanism of action for neurotensin in the ventral midbrain.     

Sex differences in densities of dopaminergic fibers and GABAergic neurons in the prenatal rat striatum.

On the basis of observations on dopaminergic neurons developing in gender-specific cultures of embryonic rat mesencephalon, we have hypothesized that as yet unknown sexual dimorphisms might be found in projection areas of dopaminergic neurons. Therefore we searched for possible sex differences in the striatum during the period when massive ingrowth of mesencephalic afferents occurs and the striatal gamma-aminobutyric acid (GABA)ergic neurons differentiate. Male and female rats of embryonic days (E) 16, 18, 20, and 21 were fixed by perfusion through the heart. Vibratome sections were cut from the striatal anlage and sequentially immunostained for GABA by the immunogold-silver technique and tyrosine hydroxylase (TH) by the avidin-biotin-peroxidase method. Ultrathin sections were scanned for numbers of GABA- and TH-immunoreactive (IR) elements. Densities of TH-IR axons as well as of GABA-IR cell body profiles progressed with time. Contacts between TH-IR axons and GABA-IR and immunonegative cells were observed as early as E-16, increasing in numbers toward later stages. Throughout prenatal development, female striata displayed higher densities of both TH-IR axon and GABA-IR cell body profiles than male ones. This is the first report of a distinct anatomical sex difference regarding two major components of a key center of motor control. Prenatal sexual differentiation of the striatum may lead to a sexually dimorphic extrapyramidal circuitry, the existence of which, in the adult, is suggested by experimental and clinical data.     

Postnatal development of striatal neurotensin immunoreactivity in relation to clusters of substance P immunoreactive neurons and the "dopamine islands" in the rat.

Conventional immunoperoxidase preparations of the coronally sectioned brains of rats killed at various times during the early postnatal period revealed the distributions of tyrosine hydroxylase, substance P, and neurotensin immunoreactivities. At birth, patches of dense tyrosine hydroxylase immunoreactivity were present across the breadth of the rostral striatum, whereas patches displaying substance P immunoreactivity were present only in its lateral half, appearing in its medial half by about postnatal day 3. Neuronal neurotensin immunoreactivity was absent in the rostral striatum at birth, although some neurotensin immunoreactive cells were present in the tail of the caudate-putamen. Rostrally, neurotensin immunoreactive cells appeared first along the lateral margin of the caudate-putamen on postnatal day 3, became numerous there about day 5, spread medially into the striatum by day 7, and achieved their medialmost distribution by about day 10. Their numbers and those of substance P immunoreactive neurons diminished thereafter. Substance P immunoreactive patches, which contained numerous labeled neurons and "puncta," shared coextensive distributions with patches of dense tyrosine hydroxylase immunoreactivity, but interdigitated with neurotensin immunoreactive cell clusters. The neurotensin immunoreactive cell clusters lacked puncta, the light microscopic representation of axon terminals, or swellings. It is concluded that the patchy infrastructure of the striatum, which is established prior to birth, is substrate for the progression of separate "waves" of elevated neuronal peptide content, one reflecting substance P and a later one reflecting neurotensin. These proceed along rostromedialward trajectories to involve interdigitating neuronal domains.     

Dopaminergic innervation of the mouse inner ear: evidence for a separate cytochemical group of cochlear efferent fibers

Immunostaining mouse cochleas for tyrosine hydroxylase (TH) and dopamine beta-hydroxylase suggests that there is a rich adrenergic innervation throughout the auditory nerve trunk and a small dopaminergic innervation of the sensory cell areas. Surgical cuts in the brainstem confirm these dopaminergic fibers as part of the olivocochlear efferent bundle. Within the sensory epithelium, TH-positive terminals are seen only in the inner hair cell area, where they intermingle with other olivocochlear terminals expressing cholinergic markers (vesicular acetylcholine transporter; VAT). Double immunostaining suggests little colocalization of TH and VAT; quantification of terminal volumes suggests that TH-positive fibers constitute only 10-20% of the efferent innervation of the inner hair cell area. Immunostaining of mouse brainstem revealed a small population of TH-positive cells in and around the lateral superior olive. Consistent with cochlear projections, double staining for the cholinergic marker acetylcholinesterase suggested that TH-positive somata are not cholinergic and vice versa. All observations are consistent with the view that a small dopaminergic subgroup of lateral olivocochlear neurons 1) projects to the inner hair cell area, 2) is distinct from the larger cholinergic group projecting there, and 3) may correspond to lateral olivocochlear "shell" neurons described by others (Warr et al. [1997] Hear. Res 108:89-111).     

Human cortical neurons contain both nicotinic and muscarinic acetylcholine receptors: an immunocytochemical double-labeling study

Using immunofluorescence histochemistry, in the human cerebral cortex neurons immunoreactive for both nicotinic and muscarinic acetylcholine receptor proteins could be demonstrated. Vibratome sections of biopsy and autopsy specimens of human temporal and occipital lobes were incubated with monoclonal antibodies specific for muscarinic (M 35) and nicotinic (WF 6) acetylcholine receptor protein. Immunoreactive sites were visualized using a biotin-streptavidin-phycoerythrin system (M 35, red fluorescence) and fluorescein-conjugated immunoglobulins (WF 6, green fluorescence). Immunofluorescence of both antibodies was preponderant in pyramidal neurons located in layers II/III and V and their apical dendrites. Some round and ovoid immunolabeled cells were encountered in layers VI and IV. About 30% of the cholinoceptive cortical neurons, in particular the pyramidal cells, displayed immunoreactivity for both receptor types. The present investigation shows a subpopulation of human cortical neurons to contain both nicotinic and muscarinic receptors. The coexistence of acetylcholine receptors may provide the morphological basis of simultaneous impact of acetylcholine on both receptor types in the same neuron of the human cerebral cortex.     

Fine structure of the nigrostriatal anlage in fetal rat brain by immunocytochemical localization of tyrosine hydroxylase

The developmental morphology and synaptic associations of neurons in the nigrostriatal anlage are examined by the electron microscopic immunocytochemical localization of tyrosine hydroxylase at embryonic (E) day 13.5 and 14.5 in rat brain. At E 13.5, immunoreactivity for the enzyme is localized throughout the cytoplasm of neuronal perikarya and processes including somatic, dendritic, and axonal growth cones. The cytoplasmic organelles in perikarya include primarily ribonucleic-protein particles, mitochondria and an immature Golgi apparatus. At E 14.5, the tyrosine hydroxylase labeled processes are detected in the lateral hypothalamus and ventrolateral caudate-putamen. The axonal processes showing immunoreactivity in the ventral mesencephalon and more rostral portions of the nigrostriatal bundle are frequently attached to unlabeled neurites by puncta adherentia. In the hypothalamus and caudate-putamen presumably transient synaptic junctions are also detected between the labeled axons and unlabeled neurons. The immature morphological features of neurons showing immunoreactivity for tyrosine hydroxylase thus indicate, that the biochemical differentiation of the nigrostriatal neurons precedes complete cytological differentiation.     

Agonist-induced internalization of corticotropin-releasing factor receptors in noradrenergic neurons of the rat locus coeruleus

Corticotropin-releasing factor (CRF) acts within the locus coeruleus (LC), to modulate activity of the LC-norepinephrine (NE) system. Combining molecular and cellular approaches, we demonstrate CRF receptor (CRFr) mRNA expression in Sprague-Dawley rat LC and provide the first in vivo evidence for agonist-induced internalization of CRFr. CRFr mRNA was detected in LC micropunches by RT-PCR. In dual labelling immunofluorescence studies, tyrosine hydroxylase (TH) containing neurons exhibited CRFr labelling. At the ultrastructural level, immunogold-silver labelling for CRFr was localized to the plasma membrane of TH-immunoperoxidase labelled dendrites. CRF (100 ng) injection into the LC produced a robust neuronal activation that peaked 10-15 min after injection and was maintained for the duration of the recording. This was associated with CRFr internalization in LC neurons that was apparent at 5 and 30 min after injection. By 5 min after injection the ratio of cytoplasmic to total dendritic CRFr-labelling was 0.81 +/- 0.01 in rats injected with CRF and 0.59 +/- 0.02 in rats injected with artificial cerebrospinal fluid (ACSF; P < 0.0001). Enhanced internalization of CRFr was maintained at 30 min after CRF injection, with the ratio being 0.86 +/- 0.02 for CRF-injected cases and 0.57 +/- 0.03 for ACSF-injected cases (P < 0.0001). Internalized CRFr was associated with early endosomes, indicative of degradation or recycling. Agonist-induced CRFr internalization in LC neurons may underlie acute desensitization to CRF or stress. This process may be a pivotal target by which stressors or pharmacological agents regulate the sensitivity of the LC-NE system to CRF and subsequent stressors.