Kirsten X. Jacobsen

Adenosine A2A and dopamine D2 heteromeric receptor complexes and their function

The existence of A2A-D2 heteromeric complexes is based on coimmunoprecipitation studies and on fluorescence resonance energy transfer and bioluminescence resonance energy transfer analyses. It has now become possible to show that A2A and D2 receptors also coimmunoprecipitate in striatal tissue, giving evidence for the existence of A2A-D2 heteromeric receptor complexes also in rat striatal tissue. The analysis gives evidence that these heteromers are constitutive, as they are observed in the absence of A2A and D2 agonists. The A2A-D2 heteromers could either be A2A-D2 heterodimers and/or higher-order A2A-D2 hetero-oligomers. In striatal neurons there are probably A2A-D2 heteromeric complexes, together with A2A-D2 homomeric complexes in the neuronal surface membrane. Their stoichiometry in various microdomains will have a major role in determining A2A and D2 signaling in the striatopallidal GABA neurons. Through the use of D2/D1 chimeras, evidence has been obtained that the fifth transmembrane (TM) domain and/or the 13 of the D2 receptor are part of the A2A-D2 receptor interface, where electrostatic epitope-epitope interactions involving the N-terminal part of 13 of the D2 receptor (arginine-rich epitope) play a major role, interacting with the carboxyl terminus of the A2A receptor. Computerized modeling of A2A-D2 heteromers are in line with these findings. It seems likely that A2A receptor-induced reduction of D2 receptor recognition, G protein coupling, and signaling, as well as the existence of A2A-D2 co-trafficking, are the consequence of the existence of an A2A-D2 receptor heteromer. The relevance of A2A-D2 heteromeric receptor complexes for Parkinson’s disease and schizophrenia is emphasized as well as for the treatment of these diseases. Finally, recent evidence for the existence of antagonistic A2A-D3 heteromeric receptor complexes in cotransfected cell lines has been summarized.

From the Golgi–Cajal mapping to the transmitter-based characterization of the neuronal networks leading to two modes of brain communication: Wiring and volume transmission ☆

After Golgi-Cajal mapped neural circuits, the discovery and mapping of the central monoamine neurons opened up for a new understanding of interneuronal communication by indicating that another form of communication exists. For instance, it was found that dopamine may be released as a prolactin inhibitory factor from the median eminence, indicating an alternative mode of dopamine communication in the brain. Subsequently, the analysis of the locus coeruleus noradrenaline neurons demonstrated a novel type of lower brainstem neuron that monosynaptically and globally innervated the entire CNS. Furthermore, the ascending raphe serotonin neuron systems were found to globally innervate the forebrain with few synapses, and where deficits in serotonergic function appeared to play a major role in depression. We propose that serotonin reuptake inhibitors may produce antidepressant effects through increasing serotonergic neurotrophism in serotonin nerve cells and their targets by transactivation of receptor tyrosine kinases (RTK), involving direct or indirect receptor/RTK interactions. Early chemical neuroanatomical work on the monoamine neurons, involving primitive nervous systems and analysis of peptide neurons, indicated the existence of alternative modes of communication apart from synaptic transmission. In 1986, Agnati and Fuxe introduced the theory of two main types of intercellular communication in the brain: wiring and volume transmission (WT and VT). Synchronization of phasic activity in the monoamine cell clusters through electrotonic coupling and synaptic transmission (WT) enables optimal VT of monoamines in the target regions. Experimental work suggests an integration of WT and VT signals via receptor-receptor interactions, and a new theory of receptor-connexin interactions in electrical and mixed synapses is introduced. Consequently, a new model of brain function must be built, in which communication includes both WT and VT and receptor-receptor interactions in the integration of signals. This will lead to the unified execution of information handling and trophism for optimal brain function and survival.

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 beta-hydroxylase (noradrenaline and/or adrenaline) to terminals positive for tyrosine hydroxylase but negative for dopamine beta-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.

Anxiolytic-like effects of the selective metabotropic glutamate receptor 5 antagonist MPEP after its intra-amygdaloid microinjection in three different non-conditioned rat models of anxiety

The intercalated islands, clusters of dopamine D1‐rich GABAergic neurons, are interposed between the basolateral and central nuclei of the amygdala, and control the traffic of nerve impulses between these two structures. Metabotropic glutamate receptor 5‐ (mGluR5)‐like immunoreactivity was studied by immunohistochemistry in this part of the amygdala and was found to be mainly restricted to the central and basolateral nuclei and to a lesser extent to the medial paracapsular intercalated islands. The role of the metabotropic glutamate receptor 5 in the modulation of anxiety has been studied in this region by microinjection of small volumes of the mGluR5 antagonist 2‐methyl‐6(phenylethenyl) pyridine (MPEP), with restricted diffusion from its injection site, into the rostral amygdala near the basolateral and central amygdaloid nuclei and the intercalated islands, and the behavior of the animals was evaluated using three non‐conditioned models of anxiety. Anxiolytic‐like effects were observed after MPEP administration in all tests used. In the White and Black Box test, MPEP (2 nmol per side) significantly increased the time spent in the white compartment of the box. In line with these results, MPEP (8 nmol per side) increased the exploration of the open arms of the Elevated Plus‐Maze. Burying behavior latency was increased and burying behavior itself was decreased in the Shock‐Probe Burying test. It is suggested that anxiolytic effects of MPEP may be mediated by blockade of mGluR5 in the basolateral and/or central amygdaloid nuclei, reducing glutamate transmission in the basolateral amygdaloid nuclei and glutamate output from the central amygdala.

Anxiolytic effects of intra-amygdaloid injection of the D1 antagonist SCH23390 in the rat.

The intercalated islands are intra-amigdaloid clusters of D1 receptor rich GABAergic neurons, which control impulse traffic between the basolateral complex and the central nucleus of the amygdala. As dopaminergic transmission within the amygdala may play a role in anxiety, the effect of the D1 antagonist SCH23390 microinjected mainly close to the rostral intercalated islands in rats was studied, using the White and Black Box test. SCH23390 reduced anxiety by an increase in the latency of the first entry into the black compartment and by an increase in the total time spent in the white compartment of the White and Black Box test, while there was no significant modification of locomotion. It is suggested that blockade of D1 receptors in the rostral intercalated islands may reduce anxiety through a reduction of GABA-mediated dishinibition of the central amygdaloid nucleus.

The distribution of dopamine D1 receptor and μ-opioid receptor 1 receptor immunoreactivities in the amygdala and interstitial nucleus of the posterior limb of the anterior commissure: Relationships to tyrosine hydroxylase and opioid peptide terminal systems

Mismatches between dopamine innervation and dopamine D1 receptor (D1) distribution have previously been demonstrated in the intercalated cell masses of the rat amygdala. Here the distribution of enkephalin and beta-endorphin immunoreactive (IR) nerve terminals with respect to their mu-opioid receptors is examined in the intercalated cell masses, along with a further immunohistochemical analysis of the dopamine/D1 mismatches. A similar analysis is also made within the extended amygdala. A spatial mismatch in distribution patterns was found between the mu-opioid receptor-1 immunoreactivity and enkephalin IR in the main intercalated island of the amygdala. Discrete cell patches of dopamine D1 receptor and mu-opioid receptor-1 IR were also identified in a distinct region of the extended amygdala, the interstitial nucleus of the posterior limb of the anterior commissure, medial division (IPACM), which displayed sparse tyrosine hydroxylase or enkephalin/beta-endorphin IR nerve terminals. Furthermore, distinct regions of the main intercalated island that showed dopamine/D1 receptor matches (the rostral and rostrolateral parts) were associated with strong dopamine and cyclic AMP regulated phosphoprotein, 32 kDa-IR in several D1 IR neuronal cell bodies and dendrites, whereas this was not the case for the dopamine/D1 mismatch areas (the rostromedial and caudal parts) of the main intercalated island. The lack of correlation between the terminal/receptor distribution patterns suggests a role for volume transmission for mu-opioid receptor- and dopamine D1 receptor-mediated transmission in distinct regions of the amygdala and extended amygdala. This may have implications for amygdaloid function, where slow long lasting responses may develop as a result of volume transmission operating in opioid peptide and dopaminergic communication.

Detection of β-endorphin in the cerebrospinal fluid after intrastriatal microinjection into the rat brain

We have investigated to what extent microinjected beta-endorphin could migrate from the rat brain parenchyma into the CSF compartment. Exogenous rat beta-endorphin (0.1 nmol) was microinjected into the left striatum 1 mm from the lateral ventricle in anesthetized male rats. CSF samples were collected at different time points up to 2 h post-injection from a catheter affixed to the atlanto-occipital membrane of the cisterna magna. Radioimmunoassay and mass spectrometry were performed on the CSF samples, and brain sections were immunostained for beta-endorphin and mu-opioid receptors. The beta-endorphin injected rats showed a marked increase in beta-endorphin immunoreactive (IR) material in the CSF, with a peak at 30-45 min post-injection, and this beta-endorphin-IR material existed mainly as the intact beta-endorphin peptide. The immunohistochemistry results revealed the appearance of distinct beta-endorphin-IR cell bodies in the globus pallidus and the bed nucleus of stria terminalis supracapsular part, regions distant from the injection site, at 2 h post-injection of exogenous beta-endorphin. The beta-endorphin-IR in several of the globus pallidus cell bodies colocalized with the mu-opioid receptor-IR at the cell surface. These findings show that upon delivery of synthetic beta-endorphin, there is a significant intracerebral spread of the injected peptide, reaching regions far from the site of injection via diffusion in the extracellular space and flow in the cerebrospinal fluid. This may be of relevance when interpreting studies based on intracerebral injections of peptides, and advances our knowledge regarding the migration of compounds within the brain.

HES1 regulates 5-HT1A receptor gene transcription at a functional polymorphism: essential role in developmental expression.

Mammalian HES1 and HES5 are abundant in developing CNS and inhibit neurogenesis, while HES6 promotes neurogenesis. An early serotonergic differentiation marker, the 5-HT1A receptor, is repressed by HES5 and DEAF1 which recognize the C(-1019), but not G(-1019) allele of a human 5-HT1A promoter polymorphism associated with mood disorders. We tested whether HES1 and HES6 regulate transcriptional activity at this element. HES1 strongly repressed 5-HT1A transcription in neuronal and non-neuronal cells, while HES6 reversed HES1- and HES5-mediated repression. Mutation of a putative HES consensus site blocked HES1 and HES5, but, unlike HES5, HES1 repressed at the G(-1019) allele. To address its role in vivo, the temporal expression of 5-HT1A receptor RNA and protein was examined in HES1-/- mice, and elevated levels in E12.5 hindbrain and midbrain were observed. Thus, HES1 and HES6 oppositely regulate 5-HT1A receptor transcription and HES1 is required for its correct developmental expression.

Differential signaling of dopamine‐D2S and ‐D2L receptors to inhibit ERK1/2 phosphorylation

Although they have distinct functions, the signaling of dopamine‐D2 receptor short and long isoforms (D2S and D2L) is virtually identical. We compared inhibitory regulation of extracellular signal‐regulated kinases (ERK1/2) in GH4 pituitary cells separately transfected with these isoforms. Activation of rat or human dopamine‐D2S, muscarinic or somatostatin receptors inhibited thyrotropin‐releasing hormone‐induced ERK1/2 phosphorylation, while the D2L receptor failed to inhibit this response. In order to address the structural basis for the differential signaling of D2S and D2L receptors, we examined the D2L‐SS mutant, in which a protein kinase C (PKC) pseudosubstrate site that is present in the D2L but not D2S receptor was converted to a consensus PKC site. In transfected GH4 cells, the D2L‐SS mutant inhibited thyrotropin‐releasing hormone‐induced ERK1/2 phosphorylation almost as strongly as the D2S receptor. A D2S‐triple mutant that eliminates PKC sites involved in D2S receptor desensitization also inhibited ERK1/2 activation. Similarly, in striatal cultures, the D2‐selective agonist quinpirole inhibited potassium‐stimulated ERK1/2 phosphorylation, indicating the presence of this pathway in neurons. In conclusion, the D2S and D2L receptors differ in inhibitory signaling to ERK1/2 due to specific residues in the D2L receptor alternatively spliced domain, which may account for differences in their function in vivo.

Role of the amygdaloid cholecystokinin (CCK)/gastrin‐2 receptors and terminal networks in the modulation of anxiety in the rat. Effects of CCK‐4 and CCK‐8S on anxiety‐like behaviour and [3H]GABA release

The amygdala plays a key role in fear and anxiety. The intercalated islands are clusters of glutamate‐responsive GABAergic neurons rich in cholecystokinin (CCK)‐2 receptors which control the trafficking of nerve impulses from the cerebral cortex to the central nucleus of amygdala. In this study, the nature of the CCK–glutamate–GABA interactions within the rat rostral amygdala, and their relevance for anxiety, were studied. CCK/gastrin‐like immunoreactive nerve terminals were found to be mainly restricted to the paracapsular intercalated islands and the rostrolateral part of the main intercalated island. Behaviourally, the bilateral microinjection of CCK‐4 (0.043–4.3 pmol/side) or CCK‐8S (4.3 pmol/side) into the rostrolateral amygdala reduced the open‐arm exploration in the elevated plus‐maze without affecting locomotion. In contrast, neither CCK‐4 nor CCK‐8S (0.043–4.3 pmol/side) had any effects in the shock‐probe burying test as compared with their saline‐treated controls. Biochemically, CCK‐4 (0.3 and 1.5 µm), unlike CCK‐8S, enhanced significantly the K+‐stimulated release of [3H]GABA from amygdala slices. These effects were fully prevented by prior superfusion of the slices with either the selective CCK‐2 receptor antagonist CR2945 (3 µm), or 6,7‐dinitroquinoxaline‐2,3(1H,4H)‐dione (DNQX), 10 µm, a glutamatergic (+/–)‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA)/kainate receptor antagonist. It is suggested that CCK modulates glutamate‐GABA mechanisms by acting on CCK‐2 receptors via volume transmission occurring at the level of the basolateral amygdaloid nucleus and/or by synaptic or perisynaptic volume transmission in the region of the rostrolateral main and paracapsular intercalated islands, resulting in subsequent disinhibition of the central amygdaloid nucleus and anxiety or panic‐like behaviour.