22 July 2010. The striatum, the initial processing region for cortical input to the basal ganglia, is of great interest to schizophrenia researchers because its neurons contain some of the densest populations of dopamine D2 receptors, the primary target of antipsychotic medications. In a new study from Harvard Medical School, Michael Higley and Bernardo Sabatini combined several optical and pharmacological methods to examine the mechanisms by which D2 receptors modulate excitatory inputs from the cortex to the striatum. They report, in the July 4 Nature Neuroscience, that D2 receptors compete with A2A adenosine receptors to control synaptic calcium influx through a mechanism that involves protein kinase A.
Crucial to motor control and to learning and memory, the striatum is mostly made of medium spiny neurons (MSNs). The spine-studded dendrites of MSNs receive extensive excitatory glutamatergic inputs from the cortex that act on NMDA-type glutamate receptors (NMDARs). Additional inputs from dopaminergic neurons in the substantia nigra (to dorsal striatum) or ventral tegmental area (to ventral striatum) synapse on the necks of these same spines or on dendritic shafts or cell bodies. Current evidence suggests that these dopaminergic synapses can differentially regulate their target neurons, depending on whether the contacted MSN expresses D1Rs or D2Rs (Surmeier et al., 2007).
All currently approved neuroleptic drugs counter the effects of D2Rs, so it is this receptor subclass that has been of most interest to schizophrenia researchers (see Dopamine Hypothesis of Schizophrenia). But corticostriatal neurons and their associated NMDARs have drawn increasing attention, fueled by hope that pre- or post-synaptic regulation of glutamate might be an alternative or complement to D2R blockade in the management of psychosis and mood disorders (e.g., Krystal et al., 2003; see also Glutamate Hypothesis of Schizophrenia).
New methods cast light on old questions
Various complexities in striatal anatomy and circuitry have hampered the use of traditional methods to determine precisely how dopaminergic neurons regulate D2R-expressing MSNs (Wickens, 2009). For instance, D1R- and D2R-expressing MSNs are morphologically and electrophysiologically indistinguishable. This complication has recently been surmounted by the development of mouse strains in which these MSN classes can be readily distinguished with green fluorescent protein (GFP).
Moreover, D2Rs are also expressed on corticostriatal terminals. Because dopamine diffuses into the extracellular space after its release from nigrostriatal neurons, it may exert pre-synaptic control over corticostriatal glutamate release (Bamford et al., 2004), in addition to post-synaptic control via D2Rs. To further complicate matters, striatal circuits are also regulated by a variety of other neuromodulators including acetylcholine, endocannabinoids, and adenosine, all of which may influence synaptic function.
These challenges make striatal circuits perfect for study with recently developed optical techniques that allow researchers to physiologically isolate and precisely control particular cell types in complex neural circuits. Such techniques lend themselves to studying brain slice preparations and, with the use of fiber optics, awake, behaving animals (see SRF meeting report and SRF related news story).
In the new study, Higley and Sabatini combined two-photon laser imaging, optogenetics, and light-induced “uncaging” of glutamate to investigate the modulatory relationships among D2Rs, A2ARs, NMDARs, and voltage-gated calcium channels (VGCCs) at single spines on MSN dendrites. These methods allowed them to sidestep many of the anatomical and physiological intricacies of the striatum. In particular, the uncaging technique, which releases glutamate just at an individual spine on the MSN side of the synapse, allowed the researchers to eliminate any contribution of presynaptic D2Rs.
In a slice preparation that included both mouse motor cortex and its striatal target region, Higley and Sabatini used two-photon laser-scanning microscopy to visualize D2R-containing MSNs while they performed whole-cell recordings. Pulses of blue light were used to activate light-sensitive channelrhodopsin-2 (ChR-2) ion channels in nearby corticostriatal axon terminals, which released glutamate, evoking excitatory post-synaptic potentials (EPSPs). Confirming previous work, when D2Rs were activated—both pre- and post-synaptically—by bathing the slice in the D2R agonist quinpirole, EPSPs were significantly reduced.
To determine the role of post-synaptic D2R binding in this diminution of EPSPs, the researchers then bathed the slice in a "caged" form of glutamate, and laser-light pulses were delivered near the dendritic spine, releasing the caged glutamate locally. Under these conditions, applying the D2R agonist to the slice did not significantly reduce the EPSP, supporting previous work suggesting that dopamine primarily exerts its effects on excitatory potentials and currents “back” at the pre-synaptic D2Rs. However, two-photon imaging of synaptic activity revealed that the D2R agonist did cause a dramatic, 50 percent reduction in Ca2+ entry into the MSN spine and dendrite during localized uncaging, highlighting a purely post-synaptic effect of D2Rs on calcium influx.
What's Ca2+ got to do with it?
To dissect the contributions of various mechanisms of Ca2+ entry into MSNs, the researchers applied a range of NMDAR and VGCC antagonists while uncaging glutamate locally to MSN synapses. They found that NMDARs and R-type VGCCs played the largest role in Ca2+ entry in these neurons. Further experiments confirmed that the primary mechanisms underlying D2R control of calcium entry into MSNs is the regulation of NMDARs, and to a lesser extent, of R-type VGCC activation.
Because hippocampal NMDARs are regulated by a protein kinase A (PKA)-dependent mechanism, the team introduced a PKA antagonist to determine whether this occurs in the striatum as well. This antagonist's effects on calcium influx appeared identical to those of D2R activation, hinting that D2Rs downregulate PKA to reduce NMDAR calcium influx. On the other hand, A2As, which are co-expressed with D2Rs on MSNs, interact positively with PKA, and have been shown to oppose the effects of D2R activation on striatal plasticity. In agreement with these data, Higley and Sabatini found that application of a selective A2A agonist canceled out the effects of D2R activation on NMDARs.
The authors conclude that co-expressed D2Rs and A2As competitively influence Ca2+ entry into MSNs via opposing effects on a PKA-based mechanism that regulates NMDARs. However, D2Rs have a limited capacity to override A2A control by a divergent PKA-independent pathway that lessens Ca2+ influx into MSNs by regulating a small subset of VGCCs.
Although they do not speculate on how these findings might relate to psychotic symptoms or treatment, the authors do make suggestions about relevance to the electrophysiological behavior of neurons, writing, "In striatopallidal neurons, activation of D2Rs during pairing of pre- and post-synaptic activity is sufficient to convert NMDAR-dependent LTP into LTD, and this switch is prevented by coactivation of A2Ars [Shen et al., 2008]. Our finding that D2R and A2AR activities bi-directionally control NMDAR-mediated Ca2+ influx provides a potential mechanism for these observations."—Pete Farley.*
Higley MJ, Sabatini BL. Competitive regulation of synaptic Ca2+ influx by D2 dopamine and A2A adenosine receptors. Nat Neurosci. 2010 Jul 4. Abstract
*Contributor Pete Farley is an employee of Yale University, where he serves as managing editor of Medicine@Yale. Michael Higley, co-author of the study discussed in this news article, recently accepted a faculty position in the department of Neurobiology of Yale.