Dopamine D2 receptors gate generalization of conditioned threat responses through mTORC1 signaling in the extended amygdala.

Overgeneralization of conditioned threat responses is a robust clinical marker of anxiety disorders. In overgeneralization, responses that are appropriate to threat-predicting cues are evoked by perceptually similar safety-predicting cues. Inappropriate learning of conditioned threat responses may thus form an etiological basis for anxiety disorders. The role of dopamine (DA) in memory encoding is well established. Indeed by signaling salience and valence, DA is thought to facilitate discriminative learning between stimuli representing safety or threat. However, the neuroanatomical and biochemical substrates through which DA modulates overgeneralization of threat responses remain poorly understood. Here we report that the modulation of DA D2 receptor (D2R) signaling bidirectionally regulates the consolidation of fear responses. While the blockade of D2R induces generalized threat responses, its stimulation facilitates discriminative learning between stimuli representing safety or threat. Moreover, we show that controlled threat generalization requires the coordinated activation of D2R in the bed nucleus of the stria terminalis and the central amygdala. Finally, we identify the mTORC1 cascade activation as an important molecular event by which D2R mediates its effects. These data reveal that D2R signaling in the extended amygdala constitutes an important checkpoint through which DA participates in the control of threat processing and the emergence of overgeneralized threat responses.

Reversal of supersensitive striatal dopamine D1 receptor signaling and extracellular signal-regulated kinase activity in dopamine-deficient mice.

Lesions of dopaminergic nigrostriatal neurons cause supersensitivity to dopamine in the striatum. Previous work has shown that such supersensitivity, an important aspect of rodent models of Parkinson's disease, is associated with anatomically abnormal patterns in the activation of extracellular signal-regulated kinase. After lesions of dopaminergic neurons, dopamine D1-receptor agonists activate extracellular signal-regulated kinase in the dorsal striatum, something not observed in intact animals. Here we used a more selective method of dopamine depletion. Dopamine-deficient mice, in which the tyrosine hydroxylase gene is specifically inactivated in dopaminergic neurons, were used to investigate dopamine D1-receptor-mediated activation of extracellular signal-regulated kinase. In wild-type mice, acute treatment with a dopamine D1-receptor agonist results in activation of extracellular signal-regulated kinase in the nucleus accumbens without activation in the dorsal striatum. In contrast, in dopamine-deficient mice, dopamine D1-receptor-agonist treatment results in activation of extracellular signal-regulated kinase not only in the nucleus accumbens, but also throughout most of the dorsal striatum. Chronic replacement of dopamine by repeated injection of L-DOPA for 36 h reverses this supersensitive extracellular signal-regulated kinase activation. This reversal displays a dorsal to ventral progression such that, by 36 h, extracellular signal-regulated kinase activation is virtually restricted to the nucleus accumbens, as in wild-type mice. The reversal of dopamine D1-receptor activation of extracellular signal-regulated kinase in dopamine-deficient mice following chronic L-DOPA treatment shows that the lack of dopamine, rather than absence of other factors secreted from dopaminergic neurons, is responsible for dopamine supersensitivity.

Coincident stimulation of convergent cortical inputs enhances immediate early gene induction in the striatum.

The effect of coincident stimulation of convergent corticostriatal inputs was analyzed by the induction of immediate early genes in striatal neurons. Cortical motor areas were stimulated through implanted electrodes in awake, behaving rats, and the induction of the mRNAs encoding the immediate early genes (IEGs) c-fos and arc was analyzed in the striatum with in situ hybridization histochemistry. In the first experiment, unilateral stimulation of the medial agranular cortex, orofacial region of the lateral agranular cortex or the forelimb region of the lateral agranular cortex resulted in IEG induction in the striatum, which was restricted to the topographically related area receiving input from the stimulated cortical area. In a second experiment, stimulation parameters were altered, including frequency, number of pulses/train, and number of trains/s. These parameters did not have a significant effect on IEG induction. Notably, in some cases, in which there was IEG induction not only in the stimulated cortical region, but also in the homologous area in the contralateral hemisphere, very robust IEG induction was observed in the striatum. In a third experiment, the orofacial regions of the lateral agranular cortex of both hemispheres were stimulated coincidently. All of these animals showed robust striatal IEG induction. This IEG induction was attenuated by pretreatment with an NMDA antagonist MK-801. In a fourth experiment, we tested whether the coincidence of bilateral cortical stimulation contributed to the efficacy of striatal IEG induction. Either alternating stimulation or coincident stimulation of non-homologous cortical regions produced significantly lower striatal IEG induction than obtained with coincident stimulation of homologous cortical areas. Enhanced striatal IEG induction occurred in indirect striatal neurons, labeled with enkephalin, but was also present in a large number of enkephalin-negative neurons, most of which are likely direct pathway neurons. These results suggest that regional and temporal convergence of cortical inputs enhances striatal IEG induction.

Molecular effects of dopamine on striatal-projection pathways.

Gene regulation studies demonstrate that dopamine differentially regulates the direct and indirect projection neurons of the striatum through their respective expression of the D1 and D2 dopamine receptors. Induction of immediate-early genes (IEGs) in striatal neurons is used to study dopamine-receptor-mediated neuronal plasticity. In the dopamine-depleted striatum there is a switch in receptor-mediated signal transduction mechanisms to produce a supersensitive form of D1- mediated neuronal plasticity. This switch is suggested to underlie dopamine-agonist-induced dyskinetic movements that develop during the treatment of Parkinson's disease.

Cocaine effects on gene regulation in the striatum and behavior: increased sensitivity in D3 dopamine receptor-deficient mice.

Central effects of psychostimulants such as cocaine are predominantly mediated by dopamine receptors. We have used mice with a targeted deletion of the D3 dopamine receptor subtype to investigate the role of this receptor in the regulation of gene expression in striatal neurons and behavior by acute and repeated treatment with cocaine (25 mg/kg). In mice lacking D3 receptors, acute administration of cocaine has more pronounced stimulatory effects on c-fos and dynorphin expression in the dorsal and ventral striatum. The behavioral response to cocaine is also increased in these mice. These findings indicate that the D3 receptor plays an inhibitory role in the action of cocaine on behavior and gene regulation in the striatum.

Dopamine-mediated gene regulation in models of Parkinson's disease.

The normal functioning of the basal ganglia is dependent on dopamine maintaining a balance between the two major output pathways of the striatum, through the D1 and D2 dopamine receptors, which have opposing effects on these pathways. Lesions of the dopamine system, such as occur in Parkinson's disease (PD), disrupt this balance. Gene regulation studies provide a measure of the cellular and molecular effects of dopamine on striatal neurons in animal models of PD. Dopamine agonists, involving selective or mixed D1 and D2 agonists, such as levodopa, are able to reverse many of the homeostatic changes induced by striatal dopamine depletion. However, following dopamine depletion, a supersensitive responsiveness of D1 striatal neurons to dopamine agonists develops, indicated by the induction of immediate early genes. The molecular and cellular mechanisms underlying the irreversibility of this supersensitive response with long-term dopamine agonist treatments may provide insights into dyskinesias that develop with long-term levodopa therapy in the treatment of PD.

Enkephalin regulates acute D2 dopamine receptor antagonist-induced immediate-early gene expression in striatal neurons.

Projection neurons of the striatum release opioid peptides in addition to GABA. Our previous studies showed that the opioid peptide dynorphin regulates that subtype of projection neurons which sends axons to the substantia nigra/entopeduncular nucleus, as indicated by an inhibitory action of dynorphin/agonists on D1 dopamine receptor-mediated immediate-early gene induction in these neurons. The other subtype of striatal projection neurons projects to the globus pallidus and contains the opioid peptide enkephalin. Here, we investigated whether enkephalin regulates the function of striatopallidal neurons, by analysing opioid effects on immediate-early gene induction by D2 dopamine receptor blockade that occurs in these neurons. Thus, the effects of systemic and intrastriatal administration of various opioid receptor agonists and antagonists on immediate-early gene expression (c-fos, zif 268) induced by the D2 receptor antagonist eticlopride were examined with in situ hybridization histochemistry. Intrastriatal infusion of enkephalin (delta and mu), but not dynorphin (kappa), receptor agonists suppressed immediate-early gene induction by eticlopride in a dose-dependent manner. This suppression was blocked by the opioid receptor antagonist naloxone, confirming the involvement of opioid receptors. Repeated treatment with D2 receptor antagonists produces increased enkephalin expression and diminished immediate-early gene inducibility in striatopallidal neurons, as well as behavioral effects that are attenuated compared to those of acute treatment (e.g., reduced akinesia). Naloxone reversed such behavioral recovery (i.e. reinstated akinesia), but did not significantly affect suppressed immediate-early gene induction. Our results indicate that enkephalin acts, via mu and delta receptors in the striatum, to inhibit acute effects of D2 receptor blockade in striatopallidal neurons. Moreover, the present findings suggest that increased enkephalin expression after repeated D2 receptor antagonist treatment is an adaptive response that counteracts functional consequences of D2 receptor blockade, but is not involved in suppressed immediate-early gene induction. Together with our earlier findings of the role of dynorphin, these results indicate that opioid peptides in the striatum serve as negative feedback systems to regulate the striatal output pathways in which they are expressed.

Lack of a role for the D3 receptor in clozapine induction of c-fos demonstrated in D3 dopamine receptor-deficient mice.

The role of the D3 dopamine receptor in mediating the effects of clozapine was analysed using in situ hybridization histochemistry to measure the induction of the immediate early gene c-fos in different brain areas of mice lacking a functional D3 dopamine receptor compared to wild type mice. Clozapine treatment (15 and 30 mg/kg, s.c.) resulted in a dose-dependent pattern of induction of c-fos messenger RNA in the striatum, accumbens and septal area, with a non-significant increase in the prefrontal cortex. There was no difference detected in any of these areas in the level of induction between mice lacking the D3 receptor (D3-/-) and wild type (D3+/+). To determine which types of neurons in the striatum and accumbens displayed clozapine (30 mg/kg) induction of c-fos messenger RNA, a double-labeling experiment was performed using a radioactive c-fos messenger RNA probe and a digoxigenin-labeled enkephalin messenger RNA probe, the latter used as a marker of D2-containing neurons. Clozapine-induced c-fos was detected in 20% of enkephalin-positive striatal neurons and 15% of enkephalin-positive accumbens neurons, and in both areas in about 10% of enkephalin-negative, putative D1 neurons, in both D3+/+ and D3-/- mice. These results demonstrate that clozapine induction of c-fos messenger RNA is not dependent on the D3 dopamine receptor subtype in the striatum or nucleus accumbens.

Local infusion of the (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione does not block D1 dopamine receptor-mediated increases in immediate early gene expression in the dopamine-depleted striatum.

Administration of selective agonists of D1 dopamine receptors increases immediate early gene expression in striatal neurons, a response which is particularly robust in the dopamine-depleted striatum. Although interactions between dopamine and glutamate receptor-mediated responses in striatal neurons have been demonstrated in a number of experimental paradigms, our previous findings indicate that N-methyl-D-aspartate antagonists do not block D1 receptor-mediated induction of immediate early genes in the dopamine-depleted striatum. In the present study, we therefore examined interactions between D1 dopamine receptors and the (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate subtypes of glutamate receptor by determining whether striatal infusion of the (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione would block D1 receptor-mediated induction of the immediate early genes c-fos and zif268 in the dopamine-depleted striatum. Striatal infusion of 6-cyano-7-nitroquinoxaline-2,3-dione (1 mM) completely blocked (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate-induced c-fos and zif268 expression. However, 6-cyano-7-nitroquinoxaline-2,3-dione (1 microM-1 mM) did not significantly affect induction of c-fos and zif268 by D1 receptor stimulation (SKF 38393, 2 mg/kg, i.p.) in the dopamine-depleted striatum. To more generally block excitatory input, tetrodotoxin (10 microM) was infused into the striatum of rats receiving a D1 agonist. Local infusion of tetrodotoxin had minimal effect on induction of c-fos and zif268 in the dopamine-depleted striatum. In contrast, tetrodotoxin abolished induction of c-fos and zif268 messenger RNAs by the D2 antagonist eticlopride (0.5 mg/kg, i.p.) in both intact rats and dopamine-depleted rats receiving continuous D2 agonist treatment (quinpirole, 0.5 mg/kg/day). The results indicate that D1 receptor-mediated induction of immediate early genes in the dopamine-depleted striatum occurs by mechanisms that are independent of excitatory input through (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptors.

Role of dynorphin and enkephalin in the regulation of striatal output pathways and behavior.

Projection neurons in the striatum give rise to two output systems, the "direct" and "indirect" pathways, which antagonistically regulate basal ganglia output. While all striatal projection neurons utilize GABA as their principal neurotransmitter, they express different opioid peptide co-transmitters and also different dopamine receptor subtypes. Neurons of the direct pathway express the peptide dynorphin and the D1 dopamine receptor, whereas indirect pathway neurons express the peptide enkephalin and the D2 receptor. In the present review, we summarize our findings on the function of dynorphin and enkephalin in these striatal output pathways. In these studies, we used D1- or D2-receptor-mediated induction of immediate-early genes as a cellular response in direct or indirect projection neurons, respectively, to investigate the role of these opioid peptides. Our results suggest that the specific function of dynorphin and enkephalin is to dampen excessive activation of these neurons by dopamine and other neurotransmitters. Levels of these opioid peptides are elevated by repeated, excessive activation of these pathways, which appears to be an adaptive or compensatory response. Behavioral consequences of increased opioid peptide function in striatal output pathways may include behavioral sensitization (dynorphin) and recovery of motor function (enkephalin).

Expression of GTPase-deficient Gialpha2 results in translocation of cytoplasmic RGS4 to the plasma membrane.

The members of a recently identified protein family termed regulators of G-protein signaling (RGS) act as GTPase-activating proteins for certain Galpha subunits in vitro, but their physiological effects in cells are uncertain in the face of similar biochemical activity and overlapping patterns of tissue expression. Consistent with its activity in in vitro GTPase-activating protein assays, RGS4 interacts efficiently with endogenous proteins of the Gi and Gq subclasses of Galpha subunits but not with G12alpha or Gsalpha. Unlike other RGS proteins such as RGS9, RGS-GAIP, and Sst2p, which have been reported to be largely membrane-associated, a majority of cellular RGS4 is found as a soluble protein in the cytoplasm. However, the expression of a GTPase-deficient Gialpha subunit (Gialpha2-Q204L) resulted in the translocation of both wild type RGS4 and a non-Gialpha-binding mutant (L159F) to the plasma membrane. These data suggest that RGS4 may be recruited to the plasma membrane indirectly by G-protein activation and that multiple RGS proteins within a given cell might be differentially localized to determine a physiologic response to a G-protein-linked stimulus.

A complex program of striatal gene expression induced by dopaminergic stimulation.

Dopamine acting in the striatum is necessary for normal movement and motivation. Drugs that change striatal dopamine neurotransmission can have long-term effects on striatal physiology and behavior; these effects are thought to involve alterations in gene expression. Using the 6-hydroxydopamine lesion model of Parkinson's disease and differential display PCR, we have identified a set of more than 30 genes whose expression rapidly increases in response to stimulation of striatal dopamine D1 receptors. The induced mRNAs include both novel and previously described genes, with diverse time courses of expression. Some genes are expressed at near-maximal levels within 30 min, whereas others show no substantial induction until 2 hr or more after stimulation. Some of the induced genes, such as CREM, CHOP, and MAP kinase phosphatase-1, may be components of a homeostatic response to excessive stimulation. Others may be part of a genetic program involved in cellular and synaptic plasticity. A very similar set of genes is induced in unlesioned animals by administration of the psychostimulant cocaine or the antipsychotic eticlopride, although in distinct striatal cell populations. In contrast to some previously described early genes, most of the novel genes are not induced in cortex by apomorphine, indicating specificity of induction. Thus we have identified novel components of a complex, coordinated genetic program that is induced in striatal cells in response to various dopaminergic manipulations.

Catecholamines in the central nervous system. Overview.

A goal of this part is to examine the functional impact of catecholaminergic systems on CNS function as it relates to normal and pathological states. The participants achieve their objectives individually by relating their high-quality work on this expansive topic, while maintaining a focus on the functional implications of their findings. Nonetheless, despite the necessarily broad nature of the topics presented, there is a remarkable degree of convergence of information. Several subthemes have emerged as a consequence of considering this work in its entirety. First is the importance of examining neurotransmitter effects, not in isolation, but in terms of interactions with other neurotransmitter systems. History has shown that a limited focus often produces confusing or inconsistent results, which become increasingly clear on consideration of the state of the organism. Second is the importance of examining pharmacological and pathophysiological interactions in light of the anatomy of the system and how developmental influences can alter this relationship. Such very general considerations have been found to provide an essential ingredient in understanding the nature of catecholamine function within this complex system.

Developmental expression of tyrosine hydroxylase, D2-dopamine receptor and substance P genes in the carotid body of the rat.

Alterations in the level of putative neurotransmitters/neuromodulators and corresponding receptors may be a possible mechanism involved in changes in chemosensitivity of peripheral chemoreceptors in the carotid body during development. Using quantitative in situ hybridization histochemistry, levels of messenger RNAs encoding tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, the D2-dopamine receptor and substance P of newborn rats at postnatal days 0, 2, 14 and 21 were determined. For comparison, during the same time points during development, we also determined the level of expression of these messenger RNAs in the cells of the superior cervical ganglion which are not chemosensitive. Tyrosine hydroxylase and D2-dopamine receptor messenger RNAs were co-localized in many of the cells in both the carotid body and the superior cervical ganglion. In the carotid body, the level of tyrosine hydroxylase messenger RNA expression was greatest at birth, significantly decreased by 48 h postnatal age and remained decreased at 14 and 21 postnatal days. In contrast, D2-dopamine receptor messenger RNA levels significantly increased with postnatal age in the carotid body. This profile of an D2-dopamine receptor was not observed in the superior cervical ganglion where tyrosine hydroxylase and D2-dopamine receptor messenger RNAs levels did not significantly change from postnatal days 0 to 21. Lastly, in the rat carotid body, substance P messenger RNA was not detected. However, substance P messenger RNA was abundant in the nodose and petrosal ganglion. The increasing contribution of carotid body on ventilation with increasing postnatal age is associated with changes in levels of gene expression for tyrosine hydroxylase and D2-dopamine receptor in the carotid body.

Dynorphin regulates D1 dopamine receptor-mediated responses in the striatum: relative contributions of pre- and postsynaptic mechanisms in dorsal and ventral striatum demonstrated by altered immediate-early gene induction.

Dynorphin, an endogenous kappa opioid receptor ligand, acts in the striatum to regulate the response of striatonigral neurons to D1 dopamine receptor stimulation. We investigated the relative contributions of both presynaptic kappa receptors on dopamine terminals and postsynaptic kappa receptors on striatal neurons by analyzing opioid regulation of D1 effects in the absence of presynaptic kappa receptors, after 6-hydroxydopamine depletion of striatal dopamine. D1-receptor-mediated immediate-early gene induction was measured by using in situ hybridization histochemistry. First, repeated treatment with the D1-receptor agonist SKF-38393 (2 mg/kg/day, 3-14 days) was used to increase dynorphin levels in rats with dopamine depletions. In the nucleus accumbens, increased dynorphin expression was accompanied by reduced induction of the immediate-early genes c-fos and zif 268 by SKF-38393. In contrast, in dorsal/lateral aspects of the dopamine-depleted striatum, this D1 response was sustained despite a large increase in dynorphin expression. These results are consistent with a requirement of dopamine terminals (presynaptic kappa receptors) for the inhibitory action of dynorphin in the dorsal/lateral striatum, but not in the ventral striatum. Second, the kappa receptor agonist spiradoline (1-10 mg/kg) reduced c-fos and zif 268 induction by SKF-39393 (2.5 mg/kg) preferentially in ventral parts of the dopamine-depleted striatum, which contain higher levels of kappa receptor mRNA and binding. These results also indicate that postsynaptic kappa receptors contribute to the inhibition of the D1 response at least in the ventral striatum. Together, these results indicate that dynorphin in the striatum functions to regulate dopamine input to striatonigral neurons, acting at both pre- and postsynaptic sites, and that the relative contributions of these mechanisms differ between dorsal and ventral striatal regions.

D1 dopamine receptor-deficient mouse: cocaine-induced regulation of immediate-early gene and substance P expression in the striatum.

Psychomotor stimulants such as cocaine alter gene expression in neurons of the striatum. Whereas many of these effects are mediated by D1 dopamine receptors, the involvement of other dopamine receptor subtypes or neurotransmitters is likely. To distinguish between these possibilities, regulation by cocaine of immediate-early genes and genes encoding neuropeptides was analysed in mice that lack functional D1 receptors. Gene expression was examined with in situ hybridization histochemistry. In these animals, cocaine failed to induce the immediate-early genes c-fos and zif 268. In contrast, substance P expression was abnormally increased by this drug. These results demonstrate that some of the effects of cocaine on gene regulation are mediated via D1 receptor-dependent mechanisms, as evidenced by the absence of immediate-early gene induction in D1-deficient mice, whereas others also involve additional, non-D1 receptor mechanisms, as shown for substance P expression.

D1 dopamine receptor-mediated induction of zif268 and c-fos in the dopamine-depleted striatum: differential regulation and independence from NMDA receptors.

Excitatory amino acid afferents from cerebral cortex and dopamine afferents from the substantia nigra synapse on common projection neurons in the striatum. Activation of D1 dopamine receptors increases immediate early gene expression in the striatum and conductance through the N-methyl-d-aspartate (NMDA) receptor. To examine the contribution of NMDA receptor activation to dopamine receptor-mediated responses, we determined the effects of intrastriatal administration of NMDA antagonists on immediate early gene expression in the striatum and rotational behavior induced by stimulation of the D1 receptor in rats with unilateral dopamine depletions. Systemic administration of SKF 38393 increased c-fos and zif268 mRNAs in the striatum and induced contralateral rotation. Intrastriatal infusion of the competitive NMDA receptor antagonist (+/-)-3-(2-carboxypiperazin-4-yl)-propyl -1-phosphonic acid caused a dose-dependent attenuation of SKF 38393-induced rotation and partially decreased c-fos mRNA expression. However, D1-mediated increases in zif268 mRNA were not affected, except by the highest concentration of antagonist used (10 mM). Another competitive antagonist, 2-amino-5-phosphonovaleric acid, had similar effects. Like the competitive antagonists, intrastriatal infusion of the non-competitive NMDA antagonist MK-801 partially decreased c-fos, but not zif268, mRNA in the area around the microdialysis probe. However, unlike competitive antagonists, local infusion of 1 mM MK-801 potentiated D1-mediated increases in c-fos and zif268 mRNAs in lateral striatum. These data suggest that 1) some D1 dopamine receptor-mediated effects on striatal function are independent of ongoing NMDA receptor activation, whereas other effects are at least partially mediated by NMDA receptor activity in the striatum, and 2) competitive and non-competitive antagonists of the NMDA receptor differently affect D1-mediated immediate early gene expression in the striatum.

A targeted mutation of the D3 dopamine receptor gene is associated with hyperactivity in mice.

While most effects of dopamine in the brain are mediated by the D1 and D2 receptor subtypes, other members of this G protein-coupled receptor family have potentially important functions. D3 receptors belong to the D2-like subclass of dopamine receptors, activation of which inhibits adenylyl cyclase. Using targeted mutagenesis in mouse embryonic stem cells, we have generated mice lacking functional D3 receptors. A premature chain-termination mutation was introduced in the D3 receptor gene after residue Arg-148 in the second intracellular loop of the predicted protein sequence. Binding of the dopamine antagonist [125I]iodosulpride to D3 receptors was absent in mice homozygous for the mutation and greatly reduced in heterozygous mice. Behavioral analysis of mutant mice showed that this mutation is associated with hyperactivity in an exploratory test. Homozygous mice lacking D3 receptors display increased locomotor activity and rearing behavior. Mice heterozygous for the D3 receptor mutation show similar, albeit less pronounced, behavioral alterations. Our findings indicate that D3 receptors play an inhibitory role in the control of certain behaviors.