Candidate Review:

Modulation of Neuronal Differentiation by Dopamine Receptors

Fazal Arain

Neuroscience Graduate Program, Vanderbilt University School of Medicine, U1205 Medical Center North, Nashville, TN 37232, USA.
Correspondence e-mail: fazal.m.arain.1@vanderbilt.edu

Abstract | Full Text | PDF

Abstract | Brain development is a prolonged process that requires a tight regulation of spatiotemporal events. Neuropsychiatric disorders, like schizophrenia and bipolar disorder have been hypothesized to have a developmental etiology despite the fact that the manifestations of the symptoms of these diseases occur later in life. It has been suggested that developmental perturbation of neuronal architecture in specific regions of the forebrain is responsible for the manifestations of these diseases. Dopamine is a neurotransmitter that appears prior to synaptogenesis and expression of the dopamine system is particularly high in the regions implicated in neuropsychiatric disorders. The receptors for dopamine are classified into two sub-families which trigger unique intracellular signaling cascades and modulation of the physiology of these receptors appear to result in permanent alteration in the morphology of dopaminoceptive neurons. A number of approaches, that include modulation of activity of these receptors by different chemical compounds, altered receptor trafficking and developmental deletion of dopamine receptors, have complemented these conclusions. It appears that modulation of neuronal morphology depends upon the type of dopamine receptor activated, the distinct intracellular signaling cascade triggered and the region of the brain that express these neurons. The cellular mechanisms involved in the modulation of neuronal morphology are still under investigation. Alterations in the intracellular calcium ion concentration upon activation of dopamine receptors could lead to the triggering of down stream signals linked to the modulation of neuronal cytoskeleton and hence neuronal morphology. Studies conducted on receptor-specific modulation of neuronal differentiation could help us better understand the pathophysiology of neuropsychiatric disorders.

Brain development is a prolonged and dynamic process that begins in the prenatal period and continues through adolescence1, 2. This phenomenon can be appreciated by looking at the examples of spatiotemporal dynamics of dendritic differentiation and synaptogenesis3. Therefore these dynamics need to be considered when studying brain development.

Both genetic and environmental factors have profound effects on the formation and integrity of brain architecture. Abnormalities in the development of the brain are linked to neurological and psychiatric disorders, despite the fact that these diseases manifest their symptoms later on in life4.  It is now believed that abnormalities in the brain architecture occur much earlier then the actual appearance of the symptoms of these diseases5. Therefore it can be postulated that the manifestations of these diseases could be the result of convergence of many different developmental pathological processes linked to these diseases6.

As the brain develops the roles played by neurotransmitters change significantly7. In the adult nervous system, neurotransmitters are primarily involved in the transmission of information across synapses8. On the one hand, they perform a much more complicated role of modulating the connectivity and subsequent function of different regions of the brain during development8. Therefore the perturbation of the biology of neurotransmitters during development could be associated with alterations in the brain architecture and consequently with neurological and psychiatric diseases.

Two areas of the brain, namely medial prefrontal cortex (MFC) and striatum (STR) have been linked to complex brain functions such as cognition9, motivation and planning etc. These two areas have also been implicated in neuropsychiatric disorders6, 10, 11. Therefore the neurotransmitter systems expressed during and after development of MFC and STR, could play a crucial role in the development of these neuropsychiatric disorders.

DOPAMINE: THE NEUROTRANSMITTER

Dopamine is a neurotransmitter that is synthesized in dopaminergic neurons from the amino acid tyrosine via a series of reactions, the rate limiting step of which is mediated by the enzyme tyrosine hydroxylase. The principle dopaminergic fiber systems in the brain include the nigrostriatal (that connects the substantia nigra pars compacta to the dorsal striatum), mesolimbic pathway (that connects ventral tegmental area to the ventral striatum) and the mesocortical pathway (that connects ventral tegmental area to the prefrontal cortex). Receptors for dopamine are expressed in particularly high concentration in two regions of the brain; namely the MFC and STR. The role of dopamine in these areas has been linked to cognition, reward, learning and movement12.

Dopamine can exert an excitatory or an inhibitory influence on its target cell by binding to the appropriate receptor subtypes. The action of dopamine is terminated by reuptake into the presynaptic neuron by the action of dopamine transporter; following which it can be either repackaged into vesicles or degraded by the enzymes monoamine oxidase and catechol-O-methyltransferase. Dopamine binds to five distinct receptors called dopamine receptor 1, 2, 3, 4 and 5 (D1-5). These are transmembrane receptors that are coupled to G protein, and hence they belong to G protein coupled receptors (GPCR) family. Based on intracellular signaling mechanisms, subtypes of the coupled G proteins and sequence of homology, dopamine receptors have been classified into two subfamilies; namely the D1-like receptor subfamily (that includes D1 and D5) and D2-like receptor subfamily (that includes D2, D3 and D4)13. D1 receptor expressing neurons co-express the neuropeptides dynorphin and substance P, while the D2 receptor expressing neurons co-express enkephlins14, 15. The D1-like receptors couple to Gαs/olf proteins and the D2-like receptors couple to Gαo/i protein. The activation of D1 and D2-like receptor subfamilies trigger intracellular signaling cascades that eventually result in accumulation and depletion of intracellular cAMP respectively. It is reported that there also exists another category of dopamine receptors, namely the D1 and D2 receptor heterodimers, that are coupled to Gαq/11 protein and are linked to the phospholipase C pathway16. The signaling transduction through the Gαq/11 has been shown to result in the release of calcium ions (Ca2+) from intracellular stores17.

It is traditionally believed that the D1 and D2 receptors are expressed by distinct neurons. Studies using insitu-hybridization techniques have shown that majority of D1 and D2 receptors are expressed on distinct neurons18. Segregation of neurons that express D1 and D2 receptor RNA has also been shown in striatum18. On the other hand evidence has been presented negating this view and introducing the idea that D1 and D2 receptor are also co-expressed on the same neurons (most probably as heterodimers). The evidence for physical interaction includes co-immunoprecipitation, western blotting and immunocytochemistry17, 19.  Evidence has also been presented by studies that used in situ hybridization20, single cell RT-PCR18, 21 and electrophysiology19 to support this model.

Studies have been conducted to define the functional characteristics of D1-D2 heterodimer expressing cells. Using HEK 293T and COS7 cells transfected with D1 and D2 cDNA, it has been shown that co-stimulation via D1 receptor agonist (SKF 81297) and D2 receptor agonist (quinpirole) can result in the stimulation of phospholipase C pathway and an increase in accumulation of intracellular Ca2+ ions17. Treatment of D1-D2 transfected HEK cells with phospholipase C inhibitor, U71322, was shown to be sufficient to deplete cytosolic Ca2+ ions22. In fact, it was shown that this Calcium signaling pathway could be blocked at different steps of the pathway, for example by Gq/11 inhibitor (YM254890), by antagonist of intracellular inositol triphosphate receptors (2-aminoethoxydiphenyl borate) and by depletion of intracellular calcium stores (by thoxydiphenyl borate)22. A calcium signal was also generated upon administration of dopamine receptor agonist SKF 83959 (that specifically stimulates the phospholipase C pathway), but it was not as strong as that generated by the co-administration of SKF 83959 and quinpirole. Interestingly, the administration of either SCH23390 (D1 receptor antagonist) or raclopride (D2 receptor antagonist) was sufficient to eliminate the signaling cascades triggered by co-administration of SKF 83959 and quinpirole22. These data support the hypothesis that D1/D2 receptor heterodimers are a distinct entity with unique activity profile.

As mentioned earlier, the dopamine system has been shown to be associated with a number of crucial physiological activities. Alterations in the dopamine system have also been implicated in neurological and psychiatric disorders including Huntington’s disease, Parkinson’s Disease, schizophrenia and bipolar disorder6, 10, 11. In fact, drugs used in the treatment of some these disorders target the dopamine system23, 24. It must also be appreciated that due to the complexities of these disorders, no single experimental model can address all aspects of these diseases.

TEMPORO-SPATIAL EXPRESSION OF DOPAMINE RECEPTORS

Dopamine is one of the neurotransmitters that appear very early in the developing brain25. In fact the expression of dopamine begins prior to synaptogenesis26. Dopaminergic neurons are born around the time of telencephalic vesicle formation, from neuroepithelial cells (the cell population from which precursor brain cells are derived)26.  It has been observed that tyrosine hydroxylase expression (a marker for dopaminergic neurons) is detectable by embryonic day 11 (E11) in mice, E12-13 in rats, E14 in rabbits and E30 in monkeys27, 28. Although the expression of dopamine is evident in the developing striatum and cortex, its expression is also seen in close proximity of the neuroepithelial cells28. This unique spatiotemporal expression of dopamine advocates its role as a modulator of neurogenesis. Although numerous studies have provided replicable evidence for the spatiotemporal expression of dopaminergic neurons in different species, the precise estimation of the dynamics of dopamine receptor expression has been a difficult task29. Unreliability of antibodies and the nonspecificity of chemical compounds that serve as dopamine receptor agonists and antagonists have contributed to this scenario29. Therefore a lot of studies have used alterative techniques such as in situ hybridization and RT PCR to define the expression pattern of dopamine receptors30, 31. Lately, using laser capture microdissection, it has been shown that the transcripts of dopamine receptors can be detected as early as E12 in mouse brain29, which coincides with the birth of striatal neurons32. It was shown that the most abundant transcript of dopamine receptor during development was of D229. It was also seen that while D1, D2 and D5 receptors show a progressive increase in expression with increasing age, D3 and D4 receptors have an oscillatory expression profile29

Following their birth, the dopaminergic neurons extend axonal branches that follow a dorsal trajectory towards the midbrain and then grow ventro-rostrally towards their targets in telencephalon, traversing the diencephalon33.  At around E17, in rats, dopaminergic axon bundles start to enter the developing striatum and some of them continue to proceed towards the cortex and eventually reach their appropriate targets33. It is also worth noting that the trajectory of growing axons and dendrites is influenced by environmental cues (molecules that bind to their appropriate receptors on growth cones; an area present at the tips of axons and dendrites). These environmental cues include ephrins, netrin, semaphorins and slits33. In a recent study, the opposing roles of netrin (that binds to DCC/ deleted in colorectal cancer) and slits (that binds to robo) in guiding dopaminergic tract was described34. Using a zebrafish model it was shown that the absence of robo2 resulted in deviation of dopaminergic axons towards midline and that this defect was partially restored upon silencing netrin expression with morpholinos in these mutants34. These opposite roles of netrin and slit signaling are consistent with in vitro models35. DCC knockout mouse model has been relatively uninformative in this regard as the homozygous mutants die soon after birth but the heterozygous mice do show an altered dopamine expression pattern in the brain and an abnormal blunt response to amphetamine treatment36.

Interestingly, modulation of D1 and D2 receptor expressed on GABAergic neurons can alter the migration of these neurons37. It was shown that D1 receptor activation caused an increase while D2 receptor activation caused a decrease in neuronal migration37. These results were also complemented with findings seen with tissue obtained from D1 and D2 receptor knock out mice37. In light of these findings it can be postulated that disruption of the dopamine system during a defined embryonic period can have significant and long lasting effects on neuronal architecture38 that have functional implications39.

In the adult brain, dopamine system orchestrates complex spatiotemporal sequence of neural events that allows information from cortex to flow to the basal ganglia and hence modulate complex functions such as motor control, cognition and behavior. The circuitry involved in the motoric function is well defined40. In short, the motor and pre-motor cortical areas send excitatory glutamatergic neuronal fibers that synapse predominantly with the medium spiny neurons of the striatum. The medium spiny neurons of striatum also receive dopaminergic input from substantia nigra pars compacta. Of these striatal medium spiny neurons, the ones that co-express substance P and D1 receptors send GABAergic fibers to globus pallidus par interna and hence become a part of the direct pathway. On the other hand medium spiny neurons that co-express D2 receptors and enkephlins send GABAergic fibers to globus pallidus par externa and hence become a part of the indirect pathway.  The direct pathway eventually allows disinhibition of its thalamic and cortical targets while indirect pathway results in their inhibition. It is becoming increasingly evident that these pathways are not only involved in motoric control but also in the control of cognitive functions40.

EVIDENCE OF DOPAMINE SYSTEM’S INVOLVEMENT IN NEURONAL DEVELOPMENT

As mentioned earlier, in light of its temporal expression pattern, dopamine appears to be a very strong candidate in modulating neuronal differentiation. In fact, there are several lines of evidence that link the dopamine system to neuronal differentiation (Figure 1).

1: Stimulation of D1 and D2 receptors has distinct consequences on neuronal morphology:  A number of studies designed to investigate the effects of modulating the activity of D1 and D2 receptors on neuronal morphology have been conducted in an in vitro model. It has been seen that the application of D1 receptor agonist, SKF 38393, causes a reduction in the extent of neurite outgrowth of neurons derived from MFC41, 42 while it causes an increase the extent of neurite outgrowth in striatal neurons43. Interestingly, these effects can be attenuated by the addition of SCH23390 (a D1 receptor blocker)41, 43. On the contrary, addition of quinpirole (a D2 receptor agonist) results in an increase in neurite outgrowth44 and branching45 of neurons derived from MFC.

Studies conducted on neuronal cell lines transfected with D2-like receptor cDNA also show an increase in neurite length and branching upon exposure to quipirole46. The activity of adenyl cyclase, as modulated by D1 and D2-like receptors, has been linked to these changes in neuronal morphology.

Figure 1 | Modulation of D1 receptor causes alteration in neuronal morphology.

D1 receptor agonist SKF83959 that signals through the Gαq/11 pathway, activates the phospholipase C pathway16. Although there is a controversy regarding the precise receptor population whose stimulation results in activation of Gαq/11 signaling cascade16, 47, it has been seen that exposure to SKF 83959 results in a robust increase in neurite outgrowth and a marked change in the morphology of neurons derived from MFC (Arain & Stanwood, unpublished observations).  Therefore it appears that the neuronal morphology of dopaminoceptive neurons is not just modulated by the type of dopamine receptor stimulated but also by the specific intracellular signaling cascade triggered.  

2: Absence of D1 receptor results in increased neurite length:  Evidence for the role of dopamine receptor in modulating neuronal differentiation has also been presented using in vivo models. As can be predicted from the in vitro model (described above) which shows that since the stimulation of D1 receptor causes a decrease in neurite outgrowth, deletion of D1 receptor should have an opposite impact. This hypothesis was supported by studies done in the D1 receptor null mouse model that showed that the dendritic morphology of MFC neurons, was long and tortuous48. These finding were not present in the visual or parietal cortices (areas receiving little dopaminergic innervation)48.

3: Cocaine exposure modulates dopamine receptor trafficking and neuronal morphology:  In utero exposure to cocaine is associated with cognitive deficit in children2. Cocaine is also an inhibitor of catecholamine transporters49. It has been shown that exposure to cocaine during a defined developmental window causes permanent changes in the morphology of dopaminoceptive neurons42. These changes include elongation of dendrites and a characteristic “wavy” morphology. These changes are also accompanied by  altered D1 receptor trafficking and uncoupling of D1 receptors to G protein42, 50. It is suggested that inhibition of dopamine transporter (by cocaine) on the presynaptic membrane results in increased concentration of dopamine in the synaptic space and this scenario is compensated by reduced surface expression of D1 receptors and their uncoupling to Gαs protein on the postsynaptic membrane. This model complements the findings seen in D1 receptor null mouse48. In both of these experimental models it is seen that the unavailability of functional D1 receptors result in an increase neurite outgrowth and a wavy morphology of MFC neurons. Therefore these findings are consistent with the hypothesis that inactivation (via mal-trafficking or deletion) or activation of D1 receptors has distinct impacts on the neuronal morphology.

MODULATION OF MICROTUBULE-ASSOCIATED PROTEIN-2 (MAP2) PHOSPHORYLATION RESULTS IN ALTERATION OF NEURITE MORPHOLOGY

As mentioned earlier, the stimulation of D1 receptors leads to intracellular accumulation of cAMP which leads to the activation of protein kinase A (PKA)23. Microtubule associated protein2 (MAP2) promotes the assembly and stabilization of microtubules51 (that holds the cytoskeletal structure intact). MAP2 activity is modulated by phosphorylation at a number of its sites by PKA52 and dephosphorylation by protein-phosphatase 2A (PP2A)53. It has been shown that one of the mechanisms involved in altering neurite length in wildtype primary neuronal cultures, following D1 receptor agonist exposure, involves the phosphorylation of amino acid residues in MAP2 by the action of PKA54. These findings are consistent with the observations seen in the in vivo model of D1 over-expressing transgenic mice54. Interestingly, a MAP2 deficient mouse model shows a decrease in microtubule density and a reduction in dendritic length of hippocampal neurons55; thus providing yet another evidence for a crucial role of MAP2 in neuronal differentiation.

ROLE OF DOWNSTREAM EFFECTORS OF Ca2+ SIGNALS IN NEURONAL MORPHOLOGY

As mentioned earlier, the modulation of dopamine receptors results in alteration of the Ca2+ signals. Therefore it is likely that Ca2+ signals modulates down stream signaling cascades that eventually results in altering neuronal morphology56. One of these down stream signals is the modulation of calcium/calmodulin-dependent protein kinase II (CamKII) activity57. It’s been shown that constitutive activity of CamKII results in inhibition of dendritic growth, while its inhibition causes an increase in dendritic growth57. Furthermore, in another study it was shown that CamKII signaling involves the a number of signaling molecules that activate MAP kinase which goes on to phosphorylate the transcription factor CREB and hence eventually stimulate the transcription of Wnt-2 which directly stimulate dendritic growth58. Hence Ca2+ signaling seems to be the key architect of neuronal morphology.

CONCLUSION

The temporo-spatial pattern of dopamine system makes it an interesting candidate to study the mechanisms of brain development. The perturbation of dopamine system results in permanent alterations in neuronal morphology, the evidence for which is provided by both in vitro and in vivo models. This perturbation appears to be modulated by a number of factors that include the expression pattern of dopamine receptor subtypes, the brain region expressing these receptors and the intracellular signaling cascades triggered by them. Alteration of neuronal morphology appears to include both the extent of neurite outgrowth and their branching pattern, which could eventually translate into alteration of neuronal circuitry. Since the changes in dopamine system appear to cause alteration in the brain architecture of regions rich in dopaminoceptive neurons (regions also implicated in the pathophysiology of neuropsychiatric disorders), the dopamine system becomes an obvious candidate to study the developmental etiology of neuropsychiatric disorders. Further studies that target both the expression pattern and the pharmacology of these receptors need to be conducted to define the effects of modulation of dopamine receptors on neuronal morphology.

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This paper describes the modulation of the phosphorylation state of MAP2 following in vitro exposure to D1 receptor agonists and how it translates into altered neuronal morphology. The authors also report  studies conducted on a D1 receptor overexpressing mouse model, that exhibit decreases in apical dendritic length of cortical pyramidal neurons and  increases in phosphorylation state of MAP2 protein.

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FURTHER INFORMATION

Gregg Stanwood’s Lab