Decrease of gap junction permeability induced by dopamine and cyclic adenosine 3':5'-monophosphate in horizontal cells of turtle retina

The axon terminals of the H1 horizontal cells of the turtle retina are electrically coupled by extensive gap junctions. Dopamine (10 nM to 10 microM) induces a narrowing of the receptive field profile of the H1 horizontal cell axon terminals, increases the coupling resistance between them, and decreases the diffusion of the dye Lucifer Yellow in the network formed by the coupled axon terminals. These actions of dopamine involve the activation of D1 receptors located on the membrane of the H1 horizontal cell axon terminals proper. Increases of the intracellular cyclic AMP concentration induced by either stimulating the adenylate cyclase activity with forskolin or inhibiting the phosphodiesterase activity with isobutylmethylxanthine, theophylline, aminophylline, or compound RO 20-1724 elicit effects similar to those of dopamine on the receptive field profile of the H1 horizontal cell axon terminals, on their coupling resistance, and on the diffusion of Lucifer Yellow in the axon terminal network. It is concluded that dopamine decreases the permeability of the gap junctions between the axon terminals of the H1 horizontal cells of the turtle retina and that this action probably involves cyclic AMP as a second messenger.     

Dopamine release from interplexiform cells in the retina: effects of GnRH, FMRFamide, bicuculline, and enkephalin on horizontal cell activity.

In teleost fish, dopaminergic interplexiform cells provide an intraretinal centrifugal pathway from the inner to the outer plexiform layer, where they make abundant synapses on cone-related horizontal cells. The interplexiform cells receive all their input in the inner plexiform layer from centrifugal fibers and amacrine cells. In fish, centrifugal fibers contain gonadotropin hormone-releasing hormone (GnRH)-like and FMRFamide-like peptides (Munz et al., 1982; Stell et al., 1984), whereas amacrine cells contain a variety of neuroactive substances, including a number of peptides. In this study, we examined the effects of GnRH, FMRFamide, bicuculline, and enkephalin on horizontal cell activity in the white perch retina in an attempt to understand the synaptic inputs to the interplexiform cells. When the retina was superfused with Ringer's solution containing GnRH, horizontal cells depolarized (approximately 10 mV), and their responses to small spots increased, whereas their responses to full-field lights decreased. Thus, GnRH closely mimicked the effects of dopamine on horizontal cells. The GnRH antagonist [D-Phe2, Pro3, D-Phe6]-GnRH blocked the effects of GnRH, as did haloperidol. GnRH also had no effect on horizontal cells in retinas treated with 6-hydroxydopamine. The results indicate that GnRH acts by stimulating the release of dopamine from interplexiform cells. FMRFamide alone produced no changes on either the membrane potential or light responses of horizontal cells, but it did suppress the effects of GnRH on horizontal cells in some experiments. FRMFamide also reversed the effects of prolonged darkness on horizontal cell responses. When bicuculline was applied to the retina, horizontal cells also depolarized (approximately 10 mV), responses to full-field illumination decreased, and responses to small spots increased. Most of the effects of bicuculline were suppressed by haloperidol, indicating that bicuculline also stimulates the release of dopamine from interplexiform cells. Similar results were obtained when [D-Ala2]-met-enkephalinamide was applied to the retina; horizontal cells depolarized (approximately 10 mV), responses to full-field stimuli decreased, and responses to the light spots increased. On the other hand, [D-Ala2]-leuenkephalinamide and [D-Ala2, D-Leu5]-enkephalin had no effects on horizontal cells. Both haloperidol and naloxone blocked the effects of [D-Ala2]-met-enkephalinamide on horizontal cells, indicating that [D-Ala2]-met-enkephalinamide stimulates dopamine release from interplexiform cells via specific opiate receptors.     

The actions of amphetamine on neurotransmitters: a brief review.

The central stimulant actions of d-amphetamine are not altered in animals in which brain stores of catecholamines have been depleted with reserpine, but they are blocked by alpha-methyltyrosine, which inhibits catecholamine synthesis. The results of a variety of experiments suggest that the central actions of amphetamine result primarily from the ability of the drug to facilitate the release of newly synthesized dopamine from nerve terminals in the forebrain. The results of experiments in animals in which dopaminergic nerve terminals in various brain regions have been selectively destroyed by intracranial microinjection of 6-hydroxydopamine reveal that the locomotor stimulant actions of relatively low doses of amphetamine are dependent upon mesolimbic dopaminergic neurons, whereas the stereotyped behaviors induced by relatively larger doses of amphetamine are dependent upon nigrostriatal dopaminergic neurons. The central actions of amphetamine appear to be the primary result of interactions with dopamine neurons, but secondarily the drug also alters the dynamics of other putative neurotransmitters (e.g. acetylcholine, 5-hydroxytryptamine) in the brain.     

Tyrosine hydroxylase immunoreactivity in the rhesus monkey retina reveals synapses from bipolar cells to dopaminergic amacrine cells

The synaptic organization of dopamine-containing amacrine cells in the rhesus monkey retina was studied using immunohistochemistry of tyrosine hydroxylase (TH), the rate-limiting enzyme in the catecholamine synthetic pathway. Cell bodies of the TH-containing neurons were primarily in the innermost tier of the inner nuclear layer. Their synaptic processes, confined to the outermost stratum of the inner plexiform layer, contained mostly small, clear vesicles and were presynaptic to unlabeled amacrine cell processes and cell bodies at junctions that were symmetrical. Synapses onto the TH-immunoreactive neurons were from bipolar cell axon terminals, nonimmunoreactive amacrine cell processes, and other TH-containing amacrine cells in a decreasing order of predominance. The bipolar cells were presynaptic to the TH-containing neuronal processes at ribbon synapses. The size, structure, and position of the bipolar cell axon terminals, which, like the TH-reactive processes, were narrowly confined to the outermost stratum of the inner plexiform layer, indicate that they are recently described giant bistratified bipolar cells. The identification of this bipolar cell input now provides evidence for a pathway from the outer plexiform layer to dopaminergic amacrine cells in the inner plexiform layer via a type of cone bipolar cell.     

Synapses from bipolar cells onto dopaminergic amacrine cells in cat and rabbit retinas

Dopaminergic amacrine cells in the vertebrate retina have long been characterized as 'interamacrine' as they were only found to be pre- and postsynaptic to other amacrine cells. Immunohistochemistry with antibodies directed against tyrosine hydroxylase (TH) revealed synapses from bipolar cell axon terminals to TH-containing neuronal processes at ribbon synapses in the rhesus monkey retina. This finding challenged the notion of the dopaminergic amacrine cell phenotype as 'interamacrine'. In order to determine if the finding of synapses from bipolar cells to dopaminergic amacrine cells could be generalized to other species, we studied the synaptic organization of dopaminergic amacrine cells in the retinas of cats and rabbits with electron microscopy of TH immunoreactivity. In both species, TH-immunoreactive processes were found to be postsynaptic to bipolar axon terminals at ribbon synapses demonstrating that the original finding in the primate may be a significant feature in the retinas of many other vertebrates as well.     

Tyrosine hydroxylase immunohistochemistry fails to demonstrate dopaminergic interplexiform cells in the turtle retina

A number of catecholaminergic, presumably dopaminergic, cells could be observed in the turtle retina, because of their tyrosine hydroxylase immunoreactivity. They were amacrine cells with a pear-shaped soma and dendrites distributed in 3 sublayers within the inner plexiform layer. Neither sclerally directed TH-positive processes nor terminals in the outer plexiform layer were observed, suggesting that dopaminergic interplexiform cells do not exist in the turtle retina.     

DARPP-32, a dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein enriched in dopamine-innervated brain regions. III. Immunocytochemical localization

Immunocytochemical studies have been carried out to determine the regional and cellular distribution of DARPP-32, a protein the phosphorylation of which can be regulated by dopamine and cAMP in intact cells. These immunocytochemical studies indicate tha DARPP-32 is localized primarily in those brain regions enriched in dopaminergic nerve terminals. Moreover, the staining pattern supports the conclusion that the DARPP-32 is present in dopaminoceptive neurons, i.e., neurons that receive a dopamine input, and that it is absent from the dopaminergic neurons themselves. Within the caudatoputamen, nucleus accumbens, olfactory tubercle, bed nucleus of the stria terminalis, and portions of the amygdaloid complex, all of which receive a strong dopamine input, DARPP-32 immunoreactivity is present in neuronal cell bodies and dendrites. In brain regions that are known to receive projections from these nuclei, puncta (presumed nerve terminals) are strongly immunoreactive for DARPP-32 but indigenous cell bodies and dendrites are not immunoreactive. These target areas include the globus pallidus, ventral pallidum, entopeduncular nucleus, and the pars reticulata of the substantia nigra. No immunoreactivity is detected in neuronal cell bodies or dendrites in any of the dopaminergic nuclei. Furthermore, nerve terminals immunoreactive for DARPP-32 do not resemble dopaminergic varicosities in either their morphology or their pattern of distribution. Many neurons are weakly immunoreactive for DARPP-32 and some of these are found in areas that apparently lack a dopaminergic input: weakly labeled neuronal cell bodies and dendrites were found throughout the neocortex, primarily in layer VI, and in the Purkinje neurons of the cerebellum. DARPP-32 immunoreactivity is also present in certain glial cells, especially in the median eminence, arcuate nucleus, and medial habenula. The present immunocytochemical studies, taken together with biochemical studies (Hemmings, H.C., Jr., A.C. Nairn, D.W. Aswad, and P. Greengard (1984) J. Neurosci. 4: 99-110; Walaas, S.I., and P. Greengard (1984) J. Neurosci. 4: 84-98) on DARPP-32, indicate that DARPP-32, is present in the subclass of dopaminoceptive neurons containing D-1 receptors (dopamine receptors coupled to adenylate cyclase). DARPP-32 may be an effective marker for certain of the actions of dopamine that are mediated through cAMP and its associated protein kinase.     

Choline acetyltransferase is found in terminals of horizontal cells that label with GABA, nitric oxide synthase and calcium binding proteins in the turtle retina

In this study, we discriminated the various types of horizontal cell in the turtle retina on their content of neuroactive substances. Double label immunocytochemistry was performed on sectioned and wholemount retina using antisera to neural- and endothelial-nitric oxide synthase (nNOS, and eNOS), calretinin (CR), calbindin (CB), gamma-aminobutyric acid (GABA) and choline acetyltransferase (ChAT). H1 cells and their axon terminals label with CR, CB and GABA. Only H1 axon terminals label with eNOS. H2 cells contain CB, CR, nNOS and GABA maybe in their dendrites. H3 cells label only with nNOS. The localization of nNOS in the H2 and H3 cells is a novel finding. None of these antibodies labels H4 cells. The photoreceptor subtypes have been differentiated by different intensity of labeling with CB. The accessory member of the double cone is less intensely labeled with CB than the principal member and rods and blue cones do not appear to label at all. ChAT-IR is located in terminal boutons of H1 and H2 horizontal cells and H1 axon terminals and these boutons contact rods and all spectral types of cones. Clearly, GABA is present in H1 horizontal cells and may be used in neurotransmission between horizontal cells and possibly for feedback pathways to photoreceptors. The evidence of nNOS immunoreactivity in H2 and H3 horizontal cells, combined with available physiological evidence, suggests that NO may be involved in electrical coupling and/or modulation of synaptic input to these types of cells. Furthermore, our results raise the possibility that cholinergic synaptic transmission may occur from horizontal cell processes to photoreceptors in the outer plexiform layer of the turtle retina.     

Neurotransmitter regulation of dopamine neurons in the ventral tegmental area

Over the last 10 years there has been important progress towards understanding how neurotransmitters regulate dopaminergic output. Reasonable estimates can be made of the synaptic arrangement of afferents to dopamine and non-dopamine cells in the ventral tegmental area (VTA). These models are derived from correlative findings using a variety of techniques. In addition to improved lesioning and pathway-tracing techniques, the capacity to measure mRNA in situ allows the localization of transmitters and receptors to neurons and/or axon terminals in the VTA. The application of intracellular electrophysiology to VTA tissue slices has permitted great strides towards understanding the influence of transmitters on dopamine cell function, as well as towards elucidating relative synaptic organization. Finally, the advent of in vivo dialysis has verified the effects of transmitters on dopamine and γ-aminobutyric acid transmission in the VTA. Although reasonable estimates can be made of a single transmitter's actions under largely pharmacological conditions, our knowledge of how transmitters work in concert in the VTA to regulate the functional state of dopamine cells is only just emerging. The fact that individual transmitters can have seemingly opposite effects on dopaminergic function demonstrates that the actions of neurotransmitters in the VTA are, to some extent, state-dependent. Thus, different transmitters perform similar functions or the same transmitter may perform opposing functions when environmental circumstances are altered. Understanding the dynamic range of a transmitter's action and how this couples in concert with other transmitters to modulate dopamine neurons in the VTA is essential to defining the role of dopamine cells in the etiology and maintenance of neuropsychiatrie disorders. Further, it will permit a more rational exploration of drugs possessing utility in treating disorders involving dopamine transmission.     

Morphological impairments in retinal neurons of the scotopic visual pathway in a monkey model of Parkinson's disease

Physiological abnormalities resulting from death of dopaminergic neurons of the central nervous system in Parkinson's disease also extend to the retina, resulting in impaired visual functions. In both parkinsonian patients and animal models, low levels of dopamine and loss of dopaminergic cells in the retina have been reported. However, the morphology and connectivity of their postsynaptic neurons, the amacrine cells, have not been analyzed. Here we report, with macaques chronically treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a model of Parkinson's disease, that morphological impairments in dopaminergic retinal neurons and their plexus in the inner retina are accompanied by an immunoreactivity decrease in gamma-aminobutyric acidergic and glycinergic amacrine cells. Especially deteriorated were AII amacrine cells, the main neuronal subtype postsynaptic to dopaminergic cells, which exhibited a marked loss of lobular appendages and dendritic processes. Concomitantly, electrical synapses among AII cells, as well as chemical synapses between these and rod bipolar cells, were highly deteriorated in parkinsonian monkeys. These results highlight that the scotopic visual pathway is severely impaired in the parkinsonian condition and provide a morphological basis for a number of abnormalities found in electrophysiological and psychophysical trials in Parkinson's disease patients and animal models.     

Intracellular cyclic-amp suppresses the permeability of gap junctions between retinal amacrine cells

Gap junctions are intercellular channels composed of subunit protein connexin and subserve electrotonic transmission between connected neurons. Retinal amacrine cells, as well as horizontal cells of the same class, are homologously connected by gap junctions. The gap junctions between these neurons extend their receptive fields, and may increase the inhibitory postsynaptic effects in the retina. In the present study, we investigated whether gap junctions between the neurons are modulated by internal messengers. The permeability of gap junctions was examined by the diffusion of intracellularly injected biotinylated tracers, biocytin or Neurobiotin, into neighboring cells since gap junctions are permeable to these molecules freely. 4% Lucifer Yellow and 6% biocytin or Neurobiotin were injected intracellularly into horizontal cells and amacrine cells in isolated retinas of carp and goldfish and Japanese dace following electrophysiological identification. In the control condition, the tracer spread into many neighboring cells from the recorded cells. Superfusion of retinas with dopamine (100 microM) suppressed diffusion of the tracer into the neighboring horizontal cells, but not in the case of amacrine cells. Intracellular injection of cyclic AMP (300 mM) completely blocked diffusion of the tracer into neighboring horizontal cells and amacrine cells. However, superfusion of retinas with 8-bromo-cyclic AMP (2 mM), membrane permeable cyclic AMP analog, permitted the tracer to diffuse into the neighboring horizontal cells or amacrine cells. Intracellular injection of cyclic GMP (300 mM) blocked the diffusion between neighboring horizontal cells, but did not suppress the diffusion between amacrine cells. These results show that the permeability of gap junctions between amacrine cells is regulated by high concentration of intracellular cyclic AMP level, but not for intracellular cyclic GMP or applied dopamine or extracellularly applied low concentrations of intracellular cyclic AMP level. The present study suggests that these laterally oriented inhibitory interneurons, horizontal cells and amacrine cells, express different connexins which may be differentially regulated by intercellular messengers.     

Rat retinal dopaminergic neurons: differential maturation of somatodendritic and axonal compartments

We examined developmental changes in dopaminergic (DA) neurons of rat pups between postnatal (P) days 3 and 21. DA cell bodies and dendrites grew progressively between P3-15. Voltage-sensitive sodium channels were present in axons at P11, but the ring-like DA axon terminals appeared only during the third postnatal week. The density of ring terminals increased markedly between P15 and P21. The vesicular monoamine transporter (VMAT2) was absent before P13 and became concentrated in DA ring terminals after P17. A steady increase in VMAT2-containing rings around AII amacrine cells occurred during the third postnatal week. The presynaptic membrane protein SNAP-25 colocalized with DA terminals, but several other presynaptic proteins tested, including synaptotagmin I, synapsin, bassoon, syntaxin, and synaptogyrin, appeared not to be associated with DA neurons. Our study shows that the somatodendritic compartment of DA neurons matures before the DA axon terminals do. Maturation of DA axons during the third postnatal week corresponds to the period of onset of visual function.     

Axon terminals containing tyrosine hydroxylase- and dopamine-beta-hydroxylase immunoreactivity form synapses with galanin immunoreactive neurons in the lateral division of the bed nucleus of the stria terminalis in the rat

Catecholaminergic projections from brainstem sources to the bed nucleus of the stria terminalis play a central role in the neurochemically mediated modulation/regulation of stress response. The lateral division of the bed nucleus of the stria terminalis (BSTL) exhibits several galanin immunoreactive (ir) neurons that are also central in the modulatory control of acute stress responses. The distribution of galaninergic nervous structures overlaps with that of the dopaminergic and noradrenergic axon terminals in the BSTL. Since both monoamines and galanin regulate/modulate the central regulatory pathways of endocrine, behavioral and physiological responses during stress, the aim of this study was to demonstrate synaptic interaction between galanin-ir nervous structures and fiber terminals immunopositive for dopamine or noradrenaline in the BSTL, thereby providing morphological data to understand better the significance of catecholamine-galanin interactions in brain areas responding to stressful stimuli. Double-labeling immunohistochemistry applied both at light and electron microscopic levels made it possible to demonstrate synaptic interactions between galanin-ir nervous structures and axon terminals immunopositive for either dopamine or noradrenaline. The dopaminergic fiber terminals innervated galanin-ir cells and dendrites in the laterodorsal division of the bed nucleus of the stria terminalis (BST), whereas the noradrenergic axons contacted galaninergic neurons and dendrites in the lateroventral BST. In this study, interactions between monoamines and galanin-ir structures were demonstrated in the BSTL which can be central in the modulatory control of the major stress regulatory pathway of the limbic-hypothalamo-pituitary-adrenal axis.     

Distribution of immunoreactivity to protein kinase C in the turtle retina

Immunocytochemical staining procedures using the HRP-complexed antibody to protein kinase C (PKC) have been carried out on the turtle retina. Wholemounts and frozen sections of retina have been studied by light microscopy to evaluate PKC immunoreactivity after stimulation of the retina with light and neurotransmitters known to be active in the vertebrate retina. The most dramatically stained sites are cone synaptic pedicles and bipolar cells under all conditions. Ganglion cells stain weakly under certain conditions. Applying the antibody to a 'control' retina under dark adapted conditions results in uniform background staining of both hyperpolarizing and depolarizing bipolar pathways, while stimulating the retina with K+ under dim light conditions results in discretely stained bipolar cells and a prominent band of staining in stratum 4 of the inner plexiform layer. Stronger stimulation of bipolar cells with their terminals contributing to strata 3 and 4 and the continuous dominant band in stratum 4 can be elicited with incubation of the retina in neurotransmitter agonists, GABA and dopamine. Incubation with dopamine, in particular, brings out the putative dopaminergic amacrine cell. The only condition in which a strong band in stratum 2 can be demonstrated is under stimulation with a flashing bar of spot of light. Thus K+ and neurotransmitter stimulation elicit PKC staining in neurons contributing to the ON or depolarizing sublamina of the IPL, while intermittent flashing light stimulus is required to elicit PKC staining in the OFF or hyperpolarizing sublamina of the IPL.     

Cholinergic axon terminals in the ventral tegmental area target a subpopulation of neurons expressing low levels of the dopamine transporter.

Cholinergic activation of dopaminergic neurons in the ventral tegmental area (VTA) is thought to play a major role in cognitive functions and reward. These dopaminergic neurons differentially project to cortical and limbic forebrain regions, where their terminals differ in levels of expression of the plasmalemmal dopamine transporter (DAT). This transporter selectively identifies dopaminergic neurons, whereas the vesicular acetylcholine transporter (VAchT) is present only in the neurons that store and release acetylcholine. We examined immunogold labeling for DAT and immunoperoxidase localization of VAchT antipeptide antisera in single sections of the rat VTA to determine whether dopaminergic somata and dendrites in this region differ in their levels of expression of DAT and/or input from cholinergic terminals. VAchT immunoreactivity was prominently localized to membranes of small synaptic vesicles in unmyelinated axons and axon terminals. VAchT-immunoreactive terminals formed almost exclusively asymmetric synapses with dendrites. Of 159 dendrites that were identified as cholinergic targets, 35% contained plasmalemmal DAT, and 65% were without detectable DAT immunoreactivity. The DAT-immunoreactive dendrites postsynaptic to VAchT-labeled terminals contained less than half the density of gold particles as seen in other dendrites receiving input only from unlabeled terminals. These results suggest selective targeting of cholinergic afferents in the VTA to non-dopaminergic neurons and a subpopulation of dopaminergic neurons that have a limited capacity for plasmalemmal reuptake of dopamine, a characteristic of those that project to the frontal cortex.     

Dopaminergic neurons in the human retina

The utilization of dopamine in the adult human retina was examined by using high-affinity uptake, localization, synthesis, and release as neurotransmitter-specific physiological probes. Autoradiographic and histochemical studies have shown that dopamine-accumulating and dopamine-containing cells of the human retina belong to a population of neurons whose somata are located in the proximal regional of the inner nuclear layer. Some of these are amacrine cells which are pre- and postsynaptic to other amacrine cells exclusively in the inner plexiform layer. However, evidence is presented which indicates the existence of interplexiform dopaminergic neurons which send processes to both plexiform layers of the retina. These neurons contain a high concentration of dopamine, take up 3H-dopamine by a hig-affinity mechanism, and release endogenous or accumulated dopamine by a Ca2+-dependent mechanism upon depolarization with high extracellular K+. An endogeneous level of about 20 pmoles dopamine per mg protein was measured in freshly isolated retina using high-pressure liquid chromatography with electrochemical detection. These results demonstrate that mechanisms for dopaminergic neurotransmission are present in the human retina.     

Autoradiographic analysis of 3H-glutamate, 3H-dopamine, and 3H-GABA accumulation in rabbit retina after kainic acid treatment

We have previously reported that exposure of isolated rabbit retina to 10(-3) M kainic acid produces profound morphological changes in specific retinal neurons (Hampton et al, 1981). We noted specific swelling of horizontal cell bodies and neurites, necrosis of cell bodies in the amacrine and ganglion cell layers, and swelling of elements in the inner plexiform layer. We now report a differential sensitivity to kainic acid of specific subclasses of amacrine cells autoradiographically labeled with 3H-glutamate, 3H-GABA, or 3H-dopamine. Three different effects were observed: (1) Labeling of neurons after incubation in 3H-glutamate was uniformly reduced while labeling of glia was much less affected. (2) The accumulation of 3H-dopamine was also decreased by kainic acid in two of the three labeled bands of the inner plexiform layer. The outermost labeled band was insensitive to kainic acid at the highest concentration tested (10(-2) M). These findings provide a basis for the subclassification of dopaminergic amacrine cells into at least two subclasses based on their sensitivity to kainic acid. (3) Kainic acid caused a dramatic increase in the labeling of GABAergic amacrine cell bodies and their terminals. This increased intensity may reflect a compensatory increase in uptake activity in response to kainic acid-induced depletion of endogenous GABA stores. These results confirm the highly toxic nature of kainic acid and demonstrate a high degree of specificity and complexity in its action in the retina.     

Synaptic organization of the dopaminergic neurons in the rabbit retina.

A number of substances were tested for their ability to label amine-accumulating neurons in the rabbit retina after fixation with OsO₄ or glutaraldehyde and OsO₄. Useful results were obtained with 5,6-dihydroxytryptamine (5,6-DHT) and 6-hydroxydopamine (6-HDA). Labelled processes were characterized by small (40–50 mm) pleomorphic synaptic vesicles containing electron-dense cores, and at times by swelling of mitochondria and by increased electron density of membranes and cytoplasm. Fluorescence microscopy showed that 5,6-DHT labelled both dopaminergic and indoleamine-accumulating neurons. In most experiments, therefore, the indoleamine-accumulating neurons were removed with 5,7-dihydroxytryptamine. In such retinas the dopaminergic processes labelled by 5,6-DHT were found to make synapses of the conventional type, characterized by an accumulation of synaptic vesicles on the presumed presynaptic side and some aggregation of material on the cytoplasmic side of the synaptic membranes and within the synaptic cleft. The dopaminergic processes were found to contact each other and also non-dopaminergic amacrine cells and their processes. Conventional synapses onto dopaminergic processes were observed from both labelled and unlabelled amacrine processes. The input from labelled neurons was observed on varicose dopaminergic processes whereas input from non-labelled elements was found on the intervaricose parts of the dopaminergic processes. No Contacts of dopaminergic processes with bipolar or ganglion cells were observed. Injections of 6-HDA gave the same results, although this drug gave less distinct labelling which made the observations less decisive than with 5,6-DHT. In retinas treated with 5,6-DHT alone (i.e., in which the indoleamine-accumulating neurons remained) numerous processes were observed which were both pre- and postsynaptic to bipolar terminals. These observations suggest that the indoleamine-accumulating processes synapse with bipolar cells. The results show that the dopaminergic neurons form a network involving only amacrine cells, suggesting a regulatory function for them. By analogy with the dopaminergic interplexiform cells of the goldfish retina, it is suggested that the dopaminergic neurons in the rabbit may regulate lateral inhibitory effects mediated by amacrine cells. Furthermore, the finding that the dopaminergic and indoleamine-accumulating cells apparently have a different synaptic organization suggests that it is appropriate to categorize amacrine cells according to their transmitter content as well as their morphology.     

In vitro analysis of the origin,migratory behavior,and maturation of cortical pyramidal cells

In cerebral cortex of rat and monkey, the neuropeptide somatostatin (SOM) marks a population of nonpyramidal cells (McDonald et al. [1982] J. Neurocytol. 11:809-824; Hendry et al. [1984] J. Neurosci. 4:2497:2517; Laemle and Feldman [1985] J. Comp. Neurol. 233:452-462; Meineke and Peters [1986] J. Neurocytol. 15:121-136; DeLima and Morrison [1989] J. Comp. Neurol. 283:212-227) that represent a distinct type of gamma-aminobutyric acid (GABA) -ergic neuron (Gonchar and Burkhalter [1997] Cereb. Cortex 7:347-358; Kawaguchi and Kubota [1997] Cereb. Cortex 7:476-486) whose synaptic connections are incompletely understood. The organization of inhibitory inputs to the axon initial segment are of particular interest because of their role in the suppression of action potentials (Miles et al. [1996] Neuron 16:815:823). Synapses on axon initial segments are morphologically heterogeneous (Peters and Harriman [1990] J. Neurocytol. 19:154-174), and some terminals lack parvalbumin (PV) and contain calbindin (Del Rio and DeFelipe [1997] J. Comp. Neurol. 342:389-408), that is also expressed by many SOM-immunoreactive neurons (Kubota et al. [1994] Brain Res. 649:159-173; Gonchar and Burkhalter [1997] Cereb. Cortex 7:347-358). We studied the innervation of pyramidal neurons by SOM neurons in rat and monkey visual cortex and examined putative contacts by confocal microscopy and determined synaptic connections in the electron microscope. Through the confocal microscope, SOM-positive boutons were observed to form close appositions with somata, dendrites, and spines of intracortically projecting pyramidal neurons of rat area 17 and pyramidal cells in monkey striate cortex. In addition, in rat and monkey, SOM boutons were found to be associated with axon initial segments of pyramidal neurons. SOM axon terminals that were apposed to axon initial segments of pyramidal neurons lacked PV, which was shown previously to label axo-axonic terminals provided by chandelier cells (DeFelipe et al. [1989] Proc. Natl. Acad. Sci. USA 86:2093-2097; Gonchar and Burkhalter [1999a] J. Comp. Neurol. 406:346:360). Electron microscopic examination directly demonstrated that SOM axon terminals form symmetric synapses with the initial segments of pyramidal cells in supragranular layers of rat and monkey primary visual cortex. These SOM synapses differed ultrastructurally from the more numerous unlabeled symmetric synapses found on initial segments. Postembedding immunostaining revealed that all SOM axon terminals contained GABA. Unlike PV-expressing chandelier cell axons that innervate exclusively initial segments of pyramidal cell axons, SOM-immunoreactive neurons innervate somata, dendrites, spines, and initial segments, that are just one of their targets. Thus, SOM neurons may influence synaptic excitation of pyramidal neurons at the level of synaptic inputs to dendrites as well as at the initiation site of action potential output.     

Dopamine-acetylcholine interaction in the rat striatum: A dual-labeling immunocytochemical study

The relationship between cholinergic neurons and dopaminergic axons in the rat striatum was examined by a dual-labeling immunocytochemical method. Cholinergic neurons were identified by their immunoreactivity for choline acetyltransferase (ChAT), and dopaminergic axon terminals were identified by their positive immunoreactivity for tyrosine hydroxylase (TH). Electron microscopic analysis of dual-labeled sections revealed that while most TH-positive terminals formed synapses with unlabeled striatal neurons and dendrites, a number of TH-positive terminals formed close appositions, highly suggestive of synapses, with both large and small dendrites as well as somata of ChAT-positive neurons. Tight appositions were also found between TH-positive terminals and ChAT-positive terminals. Moreover, TH-positive terminals and ChAT-positive terminals were found to form synapses with common dendrites of unlabeled striatal neurons. These results indicated that 1) dopaminergic axon terminals could interact directly with striatal cholinergic interneurons via tight appositions with distances comparable to conventional synapses; and 2) there is a convergence of dopaminergic and cholinergic axon terminals on noncholinergic striatal neurons.