D1 Receptor Immunoreactivity Research Articles

A role for dopamine in control of the hypoxic ventilatory response via D2 receptors in the zebrafish gill.

Dopamine is a neurotransmitter involved in oxygen sensing and control of reflex hyperventilation. In aquatic vertebrates, oxygen sensing occurs in the gills via chemoreceptive neuroepithelial cells (NECs), but a mechanism for dopamine in autonomic control of ventilation has not been defined. We used immunohistochemistry and confocal microscopy to map the distribution of tyrosine hydroxylase (TH), an enzyme necessary for dopamine synthesis, in the gills of zebrafish. TH was found in nerve fibers of the gill filaments and respiratory lamellae. We further identified dopamine active transporter (dat) and vesicular monoamine transporter (vmat2) expression in neurons of the gill filaments using transgenic lines. Moreover, TH- and dat-positive nerve fibers innervated NECs. In chemical screening assays, domperidone, a D2 receptor antagonist, increased ventilation frequency in zebrafish larvae in a dose-dependent manner. When larvae were confronted with acute hypoxia, the D2 agonist, quinpirole, abolished the hyperventilatory response. Quantitative polymerase chain reaction confirmed expression of drd2a and drd2b (genes encoding D2 receptors) in the gills, and their relative abundance decreased following acclimation to hypoxia for 48h. We localized D2 receptor immunoreactivity to NECs in the efferent gill filament epithelium, and a novel cell type in the afferent filament epithelium. We provide evidence for the synthesis and storage of dopamine by sensory nerve terminals that innervate NECs. We further suggest that D2 receptors on presynaptic NECs provide a feedback mechanism that attenuates the chemoreceptor response to hypoxia. Our studies suggest that a fundamental, modulatory role for dopamine in oxygen sensing arose early in vertebrate evolution.

Role of enteric dopaminergic neurons in regulating peristalsis of rat proximal colon.

Constipation is commonly seen in patients with Parkinson's disease associated with a loss of dopaminergic neurons in both central and enteric nervous systems. However, the roles of enteric dopaminergic neurons in developing constipation remain to be elucidated. Here, we investigated the roles of enteric dopaminergic neurons in the generation of colonic peristalsis. Cannulated segments of rat proximal colon were situated in the organ bath, abluminally perfused with physiological salt solution and luminally perfused with 0.9% saline. Drugs were applied in the abluminal solution. Changes in diameter along the length of the colonic segment were captured by a video camera and transformed into spatio-temporal maps. Fluorescence immunohistochemistry was also carried out. Blockade of nitrergic neurotransmission prevented oro-aboral propagation of peristaltic waves and caused a colonic constriction without affecting ripples, non-propagating myogenic contractions. Blockade of cholinergic neurotransmission also prevented peristaltic waves but suppressed ripples with a colonic dilatation. Tetrodotoxin (0.6μM) abolished peristaltic waves and increased ripples with a constriction. SCH 23390 (20μM), a D1 -like dopamine receptor antagonist, slowed the peristaltic waves and caused a constriction, while GBR 12909 (1μM), a dopamine reuptake inhibitor, diminished the peristaltic waves with a dilatation. Bath-applied dopamine (3μM) abolished the peristaltic waves associated with a colonic dilation in an SCH 23390 (5μM)-sensitive manner. D1 receptor immunoreactivity was co-localized to nitrergic and cholinergic neurons. Dopaminergic neurons appear to facilitate nitrergic neurons via D1 -like receptors to stabilize asynchronous contractile activity resulting in the generation of colonic peristalsis.

Isolation stress during adolescence increases conditioned place preference to cocaine in female rats

Adolescence is a developmental period characterized by neuronal growth. Environmental factors such as maternal neglect, social stress and drugs of abuse have a direct impact on future behavioral patterns. Because rats are highly social animals, we decided to investigate if isolation stress during adolescence increases conditioned place preference (CPP) to drugs of abuse. Since lower D2 receptor expression in the mesocorticolimbic system is known to increase cocaine conditioning in adult animals, and because adolescents and adults differ in dopaminergic connectivity and D2 receptor populations, we investigated if D2 receptors are altered during these conditions. Female rats were weaned at postnatal day 23 and housed singly or in pairs. At day 34 rats were tested in an open field, at day 35 in an elevated plus maze and at day 36 they were tested for CPP to cocaine (15mg/kg). At post‐natal day 48, rats were euthanized, brains collected and stored at −80°C. Single‐housed rats showed greater conditioning to cocaine when compared to group‐housed rats. This suggest that housing rats singly increases the rewarding properties of cocaine. Single‐housed rats also show lower D2 receptor immunoreactivity in the Nucleus accumbens (NAc) and the Prefrontal cortex (PFC) when compared to group‐housed rats. This correlates with the greater conditioning observed in single housed rats. Isolation stress had the effect of lowering D2 receptor populations in the NAc and PFC thus augmenting the rewarding properties of cocaine in adolescent female rats. Chronic stress during adolescence thus can lower D2 receptors and could be a determining factor in substance abuse and relapse.Support or Funding InformationFinancial assistance was provided by the Office of International Science and Engineering (OISE) of NSF through the Partnerships for Research and Education (PIRE) Program (OISE‐#1545803).

Dopamine D1 Receptor Immunoreactivity on Fine Processes of GFAP-Positive Astrocytes in the Substantia Nigra Pars Reticulata of Adult Mouse.

Substantia nigra pars reticulata (SNr), the major output nucleus of the basal ganglia, receives dopamine from dendrites extending from dopaminergic neurons of the adjacent nucleus pars compacta (SNc), which is known for its selective degeneration in Parkinson's disease. As a recipient for dendritically released dopamine, the dopamine D1 receptor (D1R) is a primary candidate due to its very dense immunoreactivity in the SNr. However, the precise location of D1R remains unclear at the cellular level in the SNr except for that reported on axons/axon terminals of presumably striatal GABAergic neurons. To address this, we used D1R promotor-controlled, mVenus-expressing transgenic mice. When cells were acutely dissociated from SNr of mouse brain, prominent mVenus fluorescence was detected in fine processes of glia-like cells, but no such fluorescence was detected from neurons in the same preparation, except for the synaptic bouton-like structure on the neurons. Double immunolabeling of SNr cells dissociated from adult wild-type mice brain further revealed marked D1R immunoreactivity in the processes of glial fibrillary acidic protein (GFAP)-positive astrocytes. Such D1R imunoreactivity was significantly stronger in the SNr astrocytes than that in those of the visual cortex in the same preparation. Interestingly, GFAP-positive astrocytes dissociated from the striatum demonstrated D1R immunoreactivity, either remarkable or minimal, similarly to that shown in neurons in this nucleus. In contrast, in the SNr and visual cortex, only weak D1R immunoreactivity was detected in the neurons tested. These results suggest that the SNr astrocyte may be a candidate recipient for dendritically released dopamine. Further study is required to fully elucidate the physiological roles of divergent dopamine receptor immunoreactivity profiles in GFAP-positive astrocytes.

Colocalization of Mating-Induced Fos and D2-Like Dopamine Receptors in the Medial Preoptic Area: Influence of Sexual Experience.

Dopamine in the medial preoptic area (mPOA) stimulates sexual activity in males. This is evidenced by microdialysis and microinjection experiments revealing that dopamine receptor antagonists in the mPOA inhibit sexual activity, whereas agonists facilitate behavior. Microdialysis experiments similarly show a facilitative role for dopamine, as levels of dopamine in the mPOA increase with mating. While the majority of evidence suggests an important role for dopamine receptors in the mPOA in the regulation of male sexual behaviors, whether sexual activity or sexual experience influence dopamine receptor function in the mPOA has not been previously shown. Here we used immunohistochemical assays to determine whether varying levels of sexual activity or experience influence the number of cells containing Fos or D2 receptor immunoreactivity. Results show that sexual experience facilitated subsequent behavior, namely experience decreased latencies. Moreover, the number of cells with immunoreactivity for Fos or D2 correlated with levels of sexual experience and sexual activity. Sexual activity increased Fos immunoreactivity. Sexually experienced animals also had significantly more D2-positive cells. Sexually inexperienced animals copulating for the first time had a larger percentage of D2-positive cells containing Fos, when compared to sexually experienced animals. Finally, regardless of experience, animals that had sex prior to sacrifice had significantly more D2-positive cells that contained Fos, vs. animals that did not copulate. These findings are noteworthy because sexually experienced animals display increased sexual efficiency. The differences in activation of D2 and changes in receptor density may play a role in this efficiency and other behavioral changes across sexual experience.

MGluR4-containing corticostriatal terminals: synaptic interactions with direct and indirect pathway neurons in mice.

Glutamatergic afferents from the cerebral cortex are the main excitatory drive of striatal projection neurons. The metabotropic glutamate receptor 4 (mGluR4) presynaptically modulates transmission at corticostriatal synapses, and is considered as a potent drug target for Parkinson's disease and other brain disorders. To better characterize the anatomical substrate that underlies the functional effects of mGluR4 in the striatum, we undertook electron microscopic localization studies of mGluR4 expression in the mouse striatum. Our data demonstrate that more than 80% mGluR4-immunoreactive structures are accounted for by unmyelinated axons and axon terminals, and that almost 50% putative glutamatergic terminals (i.e. forming asymmetric synapses) express mGluR4 in the mouse striatum. Using vGluT1 as a presynaptic marker of glutamatergic corticostriatal boutons, our findings indicate: (1) all striatal mGluR4-positive terminals co-express vGluT1 immunoreactivity, (2) 44.3% total striatal glutamatergic terminals co-express vGluT1 and mGluR4, and (3) mGluR4 is expressed in 73.4% of total striatal vGluT1-positive terminals. To determine if mGluR4 terminals target preferentially direct vs. indirect pathway neurons, mGluR4 immunostaining was combined with D1 receptor immunoreactivity. These data showed that around 30% mGluR4-immunoreactive glutamatergic terminals target D1 receptor-positive spines (i.e. direct pathway neurons), while almost 70% formed synapses with D1 receptor-negative spines (i.e. putative indirect pathway neurons). Thus, these immuno-electron microscopic studies suggest that pre-synaptic mGluR4 in striatal glutamatergic terminals is expressed almost exclusively in cortical boutons to subserve regulatory influences upon a large contingent of corticostriatal terminals that preferentially target putative "indirect" pathway striatal projection neurons in mice. These observations provide a rationale for the use of mGluR4 allosteric potentiator as a potential therapy in Parkinson's disease.

Dopamine D1 and D2 receptor immunoreactivities in the arcuate-median eminence complex and their link to the tubero-infundibular dopamine neurons.

Dopamine D1 and D2 receptor immunohistochemistry and Golgi techniques were used to study the structure of the adult rat arcuate-median eminence complex, and determine the distribution of the dopamine D1 and D2 receptor immunoreactivities therein, particularly in relation to the tubero-infundibular dopamine neurons. Punctate dopamine D1 and D2 receptor immunoreactivities, likely located on nerve terminals, were enriched in the lateral palisade zone built up of nerve terminals, while the densities were low to modest in the medial palisade zone. A codistribution of dopamine D1 receptor or dopamine D2 receptor immunoreactive puncta with tyrosine hydroxylase immunoreactive nerve terminals was demonstrated in the external layer. Dopamine D1 receptor but not dopamine D2 receptor immnunoreactivites nerve cell bodies were found in the ventromedial part of the arcuate nucleus and in the lateral part of the internal layer of the median eminence forming a continuous cell mass presumably representing neuropeptide Y immunoreactive nerve cell bodies. The major arcuate dopamine/ tyrosine hydroxylase nerve cell group was found in the dorsomedial part. A large number of tyrosine hydroxylase immunoreactive nerve cell bodies in this region demonstrated punctate dopamine D1 receptor immunoreactivity but only a few presented dopamine D2 receptor immunoreactivity which were mainly found in a substantial number of tyrosine hydroxylase cell bodies of the ventral periventricular hypothalamic nucleus, also belonging to the tuberoinfundibular dopamine neurons. Structural evidence for projections of the arcuate nerve cells into the median eminence was also obtained. Distal axons formed horizontal axons in the internal layer issuing a variable number of collaterals classified into single or multiple strands located in the external layer increasing our understanding of the dopamine nerve terminal networks in this region. Dopamine D1 and D2 receptors may therefore directly and differentially modulate the activity and/or Dopamine synthesis of substantial numbers of tubero-infundibular dopamine neurons at the somatic and terminal level. The immunohistochemical work also gives support to the view that dopamine D1 receptors and/or dopamine D2 receptors in the lateral palisade zone by mediating dopamine volume transmission may contribute to the inhibition of luteinizing hormone releasing hormone release from nerve terminals in this region.

Oxytocin reverses amphetamine-induced deficits in social bonding: evidence for an interaction with nucleus accumbens dopamine.

Drug addiction has devastating consequences on social behaviors and can lead to the impairment of social bonding. Accumulating evidence indicates that alterations in oxytocin (OT) and dopamine (DA) neurotransmission within brain reward circuitry may be involved. We investigated this possibility, as well as the therapeutic potential of OT for drug-induced social deficits, using the prairie vole (Microtus ochrogaster)-a socially monogamous rodent that forms enduring pair bonds between adult mates. We demonstrate that repeated exposure to the commonly abused psychostimulant amphetamine (AMPH) inhibits the formation of partner preferences (an index of pair bonding) in female prairie voles. AMPH exposure also altered OT and DA neurotransmission in regions that mediate partner preference formation: it decreased OT and DA D2 receptor immunoreactivity in the medial prefrontal cortex (mPFC) and nucleus accumbens (NAcc), respectively, and increased NAcc DA levels. Administration of OT directly into the mPFC of AMPH-exposed voles restored partner preferences, and altered NAcc DA levels, and this effect was dependent on OT receptor activation. Together, these data suggest that repeated AMPH exposure impairs pair bonding through an OT-mediated mechanism, and that OT and DA systems within brain reward circuitry may interact to mediate the complex relationship between drug abuse and social bonding. Further, these results provide empirical support for the idea that the central OT system may represent an important target for the treatment of social deficits in addiction.

Organic cation transporter 3 is densely expressed in the intercalated cell groups of the amygdala: Anatomical evidence for a stress hormone-sensitive dopamine clearance system

The intercalated cell groups of the amygdala (ITCs) are clusters of GABAergic neurons which exert powerful modulatory control of amygdala output, and are thought to play key roles in the extinction of conditioned fear responses. Dopamine, acting through D1 receptors, inhibits ITC neuronal activity, an action that has the potential to disinhibit amygdala activity, leading to changes in behavioral responses. Dopaminergic neurotransmission in the ITC occurs through a combination of synaptic and volume transmission. Thus, mechanisms, including transport mechanisms, that regulate extracellular dopamine concentrations in the ITC, are likely to be important determinants of amygdala function. We have recently demonstrated the expression of organic cation transporter 3 (OCT3), a high-capacity transporter for dopamine and other monoamines, throughout the rat brain. In this study, we used immunohistochemical and immunofluorescence techniques to examine the distribution of OCT3 in the ITC, to identify the phenotype of OCT3-expressing cells, and to describe the spatial relationships of OCT3 to dopaminergic terminals and dopamine D1 receptors in these areas. We observed high densities of OCT3-immunoreactive perikarya and punctae throughout the D1 receptor-rich main, anterior and paracapsular ITCs, in contrast with the basolateral amygdala, where OCT3 immunoreactive perikarya and puncta were observed at much lower density. OCT3-immunoreactive perikarya in the ITC were identified as neurons. Tyrosine hydroxylase-immunoreactive fibers in the ITC were immunonegative for OCT3, though OCT3-immunoreactive punctae were observed in close proximity to TH+ terminals. Punctate OCT3-immunoreactivity in the ITCs was observed in very close proximity (<1μm) to D1 receptor immunoreactivity. These anatomical data are consistent with the hypothesis that OCT3 plays a central role in regulating dopaminergic neurotransmission in the ITC, and that it represents a post- or peri-synaptic dopamine clearance mechanism. Inhibition of OCT3-mediated transport by corticosterone may represent a mechanism by which acute stress alters dopaminergic neurotransmission in the amygdala, leading to alterations in fear and anxiety-like behavior.

Dopamine increases Na+ absorption in the Reissner's membrane of the gerbil cochlea

The purpose of the present study was to investigate the effect of dopamine as a possible regulator of epithelial Na(+) channel (ENaC) in the Reissner's membrane (RM). RM was freshly dissected from the gerbil cochlea, and short-circuit current (Isc) was measured using the voltage-sensitive vibrating probe technique. The dopamine receptor expression was examined using immunohistochemistry. The results showed that dopamine induced activation of the amiloride-sensitive Isc, but not after pre-treatment with amiloride. The D1-like receptor antagonist SCH-23390, but not the D2-like receptor antagonist sulpiride, decreased the stimulatory effect of dopamine on RM. The effect of dopamine on Na(+) transport via ENaC was still observed after blockade of the Na(+)-K(+)-ATPase by ouabain. D1 receptor immunoreactivity was observed in RM, stria vascularis and spiral ganglion. Na(+) transport in RM is activated by dopamine possibly via D1-like receptors, and intracellular mechanisms other than cAMP-mediated pathway may be involved.

Intercalated and paracapsular cell islands of the adult rat amygdala: A combined rapid-Golgi, ultrastructural, and immunohistochemical account

The anterior and rostral paracapsular intercalated islands (AIC and PIC, respectively) were studied in the context of the amygdaloid modulation of fear/anxiety using horizontal sections. The structural analysis carried out using silver-impregnated specimens revealed that the AIC is composed of tightly packed, medium-sized spiny neurons with distinct dendritic and axonal patterns that send projecting axons to the central nucleus of the amygdala. The AIC occupies a strategic position between the basolateral amygdaloid complex and the caudal limb of the anterior commissure from which it receives fibers en passage and axon terminals. Electron microscopic observation of terminal (i.e., synaptic) degeneration 72h after the surgical interruption of the anterior commissure, confirms the synaptic interaction between the latter and the AIC neurons. These observations suggest that these islands may gate the activity of neurons from the contralateral basal forebrain and synchronize the anxiogenic output of both amygdalae. Immunohistochemical analysis indicated that, within the AIC and rostral PIC, the distance between tyrosine hydroxylase-immunoreactive terminals and the punctate dopamine D1 receptor immunoreactivity, was in the micrometer range. These results indicate a short distance and a rapid extrasynaptic form of dopamine volume transmission mediated via D1 receptors in the AIC and PIC which may enhance fear and anxiety by suppressing feed-forward inhibition in the basolateral and central amygdaloid nuclei. The strong suggestion for a commissural axon projection to the AIC documented here, coupled with the previous evidences indicting an isocortical and amygdalar contributions to the anterior commissure, opens the possibility that the AIC may be involved in decoding nerve impulses arising from both the ipsi- and contra-lateral forebrain to, in turn, modulate the homolateral amygdala.

Dopamine, vesicular transporters and dopamine receptor expression and localization in rat thymus and spleen

The localization of dopamine stores and the expression and localization of vesicular monoamine transporter (VMAT) type-1 and 2 and of dopamine D1-like and D2-like receptor subtypes were investigated in rat thymus and spleen by immunohistochemical, immunochemical techniques and by RT-PCR. In the thymus dopamine immunoreactivity was developed in the cortico-medullary junction and in the medulla, but not in the thymic cortex. In the spleen, dopamine stores were found in reticular structures in the white pulp border and in the white pulp, but not in the red one. Both thymus and spleen expressed VMAT-1 and VMAT-2 immunoreactivity as well as dopamine D1, D2, D3, D4 and D5 receptor immunoreactivity. Immunohistochemistry revealed VMAT-1, VMAT-2 and dopamine D1, D2, D3, D4 and D5 receptor immunoreactivity primarily in the thymic cortical-medulla transitional zone and to a lesser extent in the medulla but not in the cortex. In the spleen, VMAT-1, VMAT-2 and dopamine D1, D2, D3, D4 and D5 receptor immunoreactivity was located primarily in the white pulp border and to a lesser extent in the white pulp. These findings indicate that both thymus and spleen express a dopaminergic system characterized by the presence of dopamine, vesicular monoamine transporters and the five subtypes of dopamine receptors. The presence of these dopaminergic markers suggests that dopamine likely originating from immune cells and/or from sympathetic neuroeffector plexus is released in the lymphoid microenvironment. Based on the microanatomical localization of dopaminergic markers investigated, a role of dopamine in maturation and selection of lymphocytes and activation of immune responses is suggested.

The dopamine D1 receptor-rich main and paracapsular intercalated nerve cell groups of the rat amygdala: relationship to the dopamine innervation

The intercalated cell masses are GABAergic neurons interposed between the major input and output structures of the amygdala. Dopaminergic projections to the main and paracapsular intercalated islands were examined by determining the relationship of the dopamine nerve-terminal networks to the D1-receptor immunoreactive staining of cells within the intercalated islands, using double-fluorescence immunolabelling procedures in combination with confocal laser microscopy. The relationship of terminals positive for both tyrosine hydroxylase and dopamine β-hydroxylase (noradrenaline and/or adrenaline) to terminals positive for tyrosine hydroxylase but negative for dopamine β-hydroxylase (dopamine terminals) was studied in relation to the D1-receptor immunoreactivity in adjacent sections at various rostrocaudal levels. The microscopy and image analysis revealed that there was only a minor dopaminergic innervation of the D1 receptor-immunoreactive cells in the rostromedial and caudal component of the main intercalated island, suggesting volume transmission as the main communication mode for dopamine in these regions. In contrast, the D1 receptor-immunoreactive areas in the rostrolateral part of the main island and also the paracapsular intercalated islands showed a high degree of dopaminergic innervation, indicating that synaptic and perisynaptic dopamine transmission plays a dominant role in these regions. It is known that amygdala neurons are involved in the elicitation and learning of fear-related behaviors. We suggest that slow dopaminergic volume transmission in the rostromedial and caudal parts of the main intercalated island may have a role in tonic excitatory modulation in these parts of the main island, allowing GABAergic activity to develop in the central amygdaloid nucleus and thereby contributing to inhibition of fear-related behavioral and autonomic responses. In contrast, a faster synaptic and perisynaptic dopaminergic transmission in the rostrolateral part of the main intercalated island and in the paracapsular intercalated islands may have a role in allowing a more rapid elicitation of fear-related behaviors.

Dopamine Modulates Synaptic Transmission Between Rat Olfactory Bulb Neurons in Culture

The glomerular layer of the olfactory bulb (OB) contains synaptic connections between olfactory sensory neurons and OB neurons as well as connections among OB neurons. A subpopulation of external tufted cells and periglomerular cells (juxtaglomerular neurons) expresses dopamine, and recent reports suggest that dopamine can inhibit olfactory sensory neuron activation of OB neurons. In this study, whole cell electrophysiological and primary culture techniques were employed to characterize the neuromodulatory properties of dopamine on glutamatergic transmission between rat OB mitral/tufted (M/T) cells and interneurons. Immunocytochemical analysis confirmed the expression of tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, in a subpopulation of cultured neurons. D2 receptor immunoreactivity was also observed in cultured M/T cells. Dopamine reduced spontaneous excitatory synaptic events recorded in interneurons. Although the D1 receptor agonist SKF38393 and the D2 receptor agonist bromocriptine mesylate mimicked this effect, evoked excitatory postsynaptic potentials (EPSPs) recorded from monosynaptically coupled neuron pairs were attenuated by dopamine and bromocriptine but not by SKF38393. Neither glutamate-evoked currents nor the membrane resistance of the postsynaptic interneuron were affected by dopamine. However, evoked calcium channel currents in the presynaptic M/T cell were diminished during the application of either dopamine or bromocriptine, but not SKF38393. Dopamine suppressed calcium channel currents even after nifedipine blockade of L-type channels, suggesting that inhibition of the dihydropyridine-resistant high-voltage activated calcium channels implicated in transmitter release may mediate dopamine's effects on spontaneous and evoked synaptic transmission. Together, these data suggest that dopamine inhibits excitatory neurotransmission between M/T cells and interneurons via a presynaptic mechanism.

Local Application of Dopamine Inhibits Pyramidal Tract Neuron Activity in the Rodent Motor Cortex

Cortical neurons respond in a variety of ways to locally applied dopamine, perhaps because of the activation of different receptors within or among subpopulations of cells. This study was conducted to assess the effects of dopamine and the receptor subtypes that mediate the responses of a specific population of neurons, the pyramidal tract neurons (PTNs) in the rodent motor cortex. The specific subfamilies of dopamine receptors expressed by PTNs also were determined. PTNs were identified by antidromic stimulation in intact animals. Extracellular recordings of their spontaneous activity and glutamate-induced excitation were performed with multi-barrel pipettes to allow simultaneous recording and iontophoresis of several drugs. Prolonged (30 s) application of dopamine caused a progressive, nonlinear decrease in spontaneous firing rates for nearly all PTNs, with significant reductions from baseline spontaneous activity (71% of baseline levels) occurring between 20 and 30 s of iontophoresis. The D1 selective (SCH23390) and the D2 selective (eticlopride) antagonists were both effective in blocking dopamine-induced inhibition in nearly all PTNs. Mean firing levels were maintained within 3% of baseline levels during co-application of the D1 antagonist with dopamine and within 11% of baseline levels during co-application of the D2 antagonist and dopamine. SCH23390 was ineffective however, in 2 of 16 PTNs, and eticlopride was ineffective in 3 PTNs. The dopamine blockade by both antagonists in most neurons, along with the selective blockade by one, but not the other antagonist in a few neurons indicate that the overall population of PTNs exhibits a heterogeneous expression of dopamine receptors. The firing rate of PTNs was significantly enhanced by iontophoresis of glutamate (mean = 141% of baseline levels). These increases were attenuated significantly (mean= 98% of baseline) by co-application with dopamine in all PTNs, indicating dopaminergic interactions with glutamate transmission. The expression of dopamine receptors was studied with dual-labeling techniques. PTNs were identified by retrograde labeling with fast blue and the D1a, D2, or D5 receptor proteins were stained immunohistochemically. Some, but not all PTNs, showed labeling for D1a, D2, or D5 receptors. The D1a and D2 receptor immunoreactivity was observed primarily in the somata of PTNs, whereas D5 immunoreactivity extended well into the apical dendrites of PTNs. In accordance with findings of D1 and D2 receptor antagonism of dopamine's actions, the identification of three DA receptor subtypes on PTNs suggests that dopamine can directly modulate PTN activity through one or more receptor subtypes.

Region-specific targeting of dopamine D2-receptors and somatodendritic vesicular monoamine transporter 2 (VMAT2) within ventral tegmental area subdivisions.

Throughout the ventral tegmental area (VTA), dopamine is packaged within subcellular organelles by the vesicular monoamine transporter-2 (VMAT2). Somatodendritically released dopamine in this region binds to the D2 receptor (D2R) to modulate ongoing neurotransmission. Although autoregulation of mesocortical dopaminergic neurons in the parabrachial VTA (PB-VTA) is known to be less efficacious than that of mesolimbic dopaminergic neurons in the paranigral (PN-VTA), the cellular basis for this regional heterogeneity is not known. For this reason, we used electron microscopic immunocytochemistry to determine the subcellular localization of the dopamine storage vesicles (identified by the presence of VMAT2) in relation to the D2R in these VTA subdivisions. In both regions, D2R immunoreactivity was principally located on extrasynaptic dendritic plasma membranes near excitatory-type synapses. Equivalent percentages (72 and 74%) of the D2R-labeled dendrites in each region contained VMAT2-immunoreactive tubulovesicles. Of the total VMAT2-labeled dendrites, however, a significantly lower percentage in the PB-VTA (26%) than in the PN-VTA (38%) contained D2R labeling. In contrast, a significantly higher number of VMAT2 immunogold-silver deposits was seen within individual dendrites in the PB-VTA than in PN-VTA. In both regions, D2R immunoreactivity was also detected in VMAT2-negative axon terminals that formed synapses on dendrites containing VMAT2. Our results are the first to demonstrate that within VTA neurons and their afferents the D2R is strategically positioned for activation by dopamine released from dendritic storage vesicles. These findings also suggest that the potential for D2R activation may affect the expression levels of VMAT2 in VTA dendrites.

Dopamine D2-like receptors on human peripheral blood lymphocytes: a radioligand binding assay and immunocytochemical study.

1. Peripheral blood lymphocytes express dopamine D1-like and D2-like receptors which were investigated using radioligand binding assay and molecular biology techniques. Analysis of dopamine D2-like receptors expressed by human peripheral blood lymphocytes with radioligand binding assay may offer a rapid technique for assessing receptor changes in disorders characterized by involvement of the dopaminergic system. However, the suitability of radioligand binding assay techniques to measure dopamine D2-like receptors is questioned. 2. In view of the discrepancy between data of dopamine D2-like receptor determination with molecular biology and radioligand binding assay techniques, we have assayed dopamine D2-like receptors expressed by human peripheral blood lymphocytes using as radioligands the dopamine receptor agonist 7-[3H]-hydroxy-N,N-di-n-propyl-2-aminotetraline ([3H]-7-OH-DPAT) and two antagonists ([3H]-spiperone and [3H]-nemonapride). 3. Analysis of saturation curves revealed a concentration-dependent binding of all compounds to human peripheral blood lymphocytes. Dissociation constant (Kd) values averaged between 0.15 and 0.40 nM for different radioligands. The maximum density of binding sites (Bmax) was low, ranging from 4.15 +/- 0.05 fmol/10(6) cells with [3H]-spiperone and 8.66 +/- 0.04 fmol/10(6) cells with [3H]-7-OH-DPAT. 4. Displacement curves of [3H]-7-OH-DPAT, [3H]-spiperone and [3H]-nemonapride binding to human peripheral blood lymphocytes revealed, using radioligand concentrations giving the highest specific:non-specific binding ratio, a pharmacological profile consistent with the labelling of dopamine D2-like receptors. The use of higher radioligand concentrations resulted in a poorly displaceable and characterizable binding. 5. Detection of dopamine D2, D3 and D4 receptor immunoreactivity in cytospin centrifuged peripheral blood lymphocytes revealed dopamine D3 and D4 but not D2 receptor immunostaining. 6. The above findings indicate in agreement with molecular biology studies, that dopamine D2-like receptors expressed by human peripheral blood lymphocytes belong to the D3 and D4 receptor subtypes. These receptors are detectable using either dopamine D2-like receptor agonists and antagonists as radioligands if controlled experimental conditions are followed. The standardisation of immunocytochemical techniques for detecting human peripheral blood lymphocyte dopamine receptors may contribute to clarify their role in lymphocyte function or as a peripheral marker of the status of the dopaminergic system.

Postnatal development of dopamine D1 receptor immunoreactivity in the rat retina

Dopamine, an important neuromodulator in the retina, controls the balance of rod cone photoreceptor activity and influences the activity of several interneurons. The postnatal development of dopaminergic neurons, visualized immunocytochemically, was compared to the development of dopamine D1 receptor immunoreactivity. Expression of D1 receptors was monitored throughout the postnatal development of the rat retina using a subtype-specific monoclonal antibody. D1 receptors are expressed in the inner plexiform layer beginning at birth. Labeling of the inner plexiform layer changed from a diffuse pattern, staining the entire layer, to the typical adult punctate staining, that was organized in layered bands and occurred in the second postnatal week. The staining did not co-localize with dopaminergic cells; instead, it co-localized with cells in the inner nuclear layer or the ganglion cell layer. Within these cells, D1 receptors were most heavily expressed in processes stratifying in the inner plexiform layer. Staining in the outer plexiform layer and in horizontal cells was found beginning in the second postnatal week. Clustering of the D1 receptor within plexiform layers, a process typical for the well-described function of dopamine modulation in the adult, occurred late in postnatal development. A possible function of D1 receptors in neuronal development is discussed. J. Neurosci. Res. 56:397–404, 1999. © 1999 Wiley-Liss, Inc.

Postnatal development of dopamine D1 receptor immunoreactivity in the rat retina.

Dopamine, an important neuromodulator in the retina, controls the balance of rod cone photoreceptor activity and influences the activity of several interneurons. The postnatal development of dopaminergic neurons, visualized immunocytochemically, was compared to the development of dopamine D1 receptor immunoreactivity. Expression of D1 receptors was monitored throughout the postnatal development of the rat retina using a subtype-specific monoclonal antibody. D1 receptors are expressed in the inner plexiform layer beginning at birth. Labeling of the inner plexiform layer changed from a diffuse pattern, staining the entire layer, to the typical adult punctate staining, that was organized in layered bands and occurred in the second postnatal week. The staining did not co-localize with dopaminergic cells; instead, it colocalized with cells in the inner nuclear layer or the ganglion cell layer. Within these cells, D1 receptors were most heavily expressed in processes stratifying in the inner plexiform layer. Staining in the outer plexiform layer and in horizontal cells was found beginning in the second postnatal week. Clustering of the D1 receptor within plexiform layers, a process typical for the well-described function of dopamine modulation in the adult, occurred late in postnatal development. A possible function of D1 receptors in neuronal development is discussed.

Synaptic Connections Between Spiny Neurons of the Direct and Indirect Pathways in the Neostriatum of the Rat: Evidence from Dopamine Receptor and Neuropeptide Immunostaining

The flow of cortical information through the basal ganglia occurs through the so-called 'direct pathway' and 'indirect pathways'. The object of the present work was to attempt to determine whether spiny neurons in the neostriatum that give rise to the direct pathway (i.e. the striatonigral/entopeduncular pathway) and those giving rise to the indirect pathways (i.e. striatopallidal pathway) are synaptically interconnected. The approach was to carry out double immunocytochemistry at the electron microscopic level using antibodies against peptides or dopamine receptor subtypes that are selectively associated with the neurons that give rise to the direct (substance P or D1 receptors) and indirect pathways (enkephalin or D2 receptors). Sections of perfuse-fixed rat neostriatum were immunostained to reveal both substance P immunoreactivity and D2 receptor immunoreactivity or enkephalin and D1 receptor immunoreactivity, respectively. Double peroxidase methods were employed using different chromogens that were distinguishable at both the light and electron microscopic levels. In the electron microscope substance P-immunoreactive terminals were seen in synaptic contact with dendritic structures that displayed immunoreactivity for D2 receptor. Similarly, enkephalin-immunoreactive terminals were seen in synaptic contact with D1-immunoreactive dendritic structures. Thus, axon collaterals of neurons giving rise to the direct pathway form synaptic contacts with neurons that give rise to the indirect pathway and axon collaterals of neurons giving rise to the indirect pathway form synaptic contact with neurons that give rise to the direct pathway. These results indicate that the two pathways of information flow through the basal ganglia are synaptically linked at the level of the neostriatum. spiny neurons, direct pathway,indirect pathways, rat neostriatum