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Illuminating the Striatum: Optical Techniques Help Clarify the Role of D2 Receptors

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.*

Reference:
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.

Comments on News and Primary Papers


Primary Papers: Competitive regulation of synaptic Ca2+ influx by D2 dopamine and A2A adenosine receptors.

Comment by:  Philip Seeman (Disclosure)
Submitted 12 July 2010
Posted 12 July 2010

There are multiple pathways to psychosis and schizophrenia, all associated with dopamine supersensitivity and elevated amounts of D2High receptors (Seeman et al., 2005; Seeman et al., 2006; Seeman, 2008; Seeman, 2010; Seeman, 2009). The present paper by Higley and Sabatini is consistent with the multiple-pathway hypothesis. They show that the dopamine D2 receptor, which is the main target for antipsychotic drugs (Seeman et al., 1975; Seeman et al., 1976), regulates the multiple pathways of glutamate and adenosine transmission. Their work is also consistent with the fact that a glutamate agonist can treat schizophrenia (Patil et al., 2007), especially when such a glutamate drug can stimulate glutamate receptors and partly inhibit dopamine D2High receptors (Seeman, 2008; Seeman, 2009; Seeman and Guan, 2009).

References:

Seeman P, Weinshenker D, Quirion R, Srivastava LK, Bhardwaj SK, Grandy DK, Premont RT, Sotnikova TD, Boksa P, El-Ghundi M, O'dowd BF, George SR, Perreault ML, Männistö PT, Robinson S, Palmiter RD, Tallerico T. Dopamine supersensitivity correlates with D2High states, implying many paths to psychosis. Proc Natl Acad Sci U S A. 2005;102:3513-8. Abstract

Seeman P, Schwarz J, Chen JF, Szechtman H, Perreault M, McKnight GS, Roder JC, Quirion R, Boksa P, Srivastava LK, Yanai K, Weinshenker D, Sumiyoshi T. Psychosis pathways converge via D2High dopamine receptors. Synapse. 2006;60:319-46. Abstract

Seeman P. All psychotic roads lead to increased D2High dopamine receptors. A perspective. Clinical Schizophrenia & Related Psychoses. 2008 Jan;351-5.

Seeman P. All roads to schizophrenia lead to dopamine supersensitivity and elevated dopamine D2High receptors. CNS Neuroscience & Therapeutics. Epub 2010 June 18. Abstract

Seeman P. Schizophrenia model of elevated D2High receptors: Haloperidol reverses the amphetamine-induced elevation in dopamine D2High. Schizophr Res. 2009;91:191-2. Abstract

Seeman P, Chau-Wong M, Tedesco J, Wong K Brain receptors for antipsychotic drugs and dopamine: Direct binding assays. Proc Natl Acad Sci U S A. 1975;72:4376-80. Abstract

Seeman P, Lee T, Chau-Wong M, Wong K. Antipsychotic drug doses and neuroleptic/dopamine receptors. Nature. 1976;261:717-9. Abstract

Patil ST, Zhang L, Martenyi F, Lowe SL, Jackson KA, Andreev BV, Avedisova AS, Bardenstein LM, Gurovich IY, Morozova MA, Mosolov SN, Neznanov NG, Reznik AM, Smulevich AB, Tochilov VA, Johnson BG, Monn JA, Schoepp DD. Activation of mGlu2/3 receptors as a new approach to treat schizophrenia: a randomized Phase 2 clinical trial. Nat Med. 2007 Sep;13(9):1102-7. Epub 2007 Sep. 2. Erratum in: Nat Med. 2007 Oct;13(10):1264. Abstract

Seeman P. Glutamate agonists for schizophrenia stimulate dopamine D2High receptors. Schizophr Res. 2008;99(1-3):373-4. Abstract

Seeman P. Glutamate agonists for treating schizophrenia have affinity for dopamine D2High and D3 receptors. Synapse. 2009;63(8):705-9. Abstract

Seeman P, Guan H-C. Glutamate agonist LY 404,039 for treating schizophrenia has affinity for the dopamine D2High receptor. Synapse. 2009;63:935-9. Abs

View all comments by Philip Seeman

Primary Papers: Competitive regulation of synaptic Ca2+ influx by D2 dopamine and A2A adenosine receptors.

Comment by:  Jose Bargas
Submitted 6 September 2010
Posted 16 September 2010
  I recommend this paper

In a recent study by Kravitz et al., 2010, it is shown, also with optogenetic techniques (channelrhodopsin expressed in direct or indirect medium spiny neurons—MSNs—of transgenic mice), that specific stimulation of D2R-expressing indirect pathway MSNs (with an implanted laser) causes akinesia and rigidity in the animals as long as the laser stimulus is on. Once the illumination is turned off, the behavior returns to normal. Moreover, they show that stimulation of D1R-expressing direct pathway MSNs cause the opposite: hyperkinesia and hyperactivity. Furthermore, if a partial parkinsonism is induced in these mice, direct pathway stimulation rescues the animals from the pathological condition. These experiments give strong support to the basal ganglia pathophysiological model launched by Albin et al. (1989) more than 20 years ago. This model is based in the following assumptions: 1) dopamine acting on D1R stimulates direct pathway MSNs firing, and that facilitates movement; 2) dopamine acting on D2R represses indirect pathway MSNs, and that facilitates movement. Either D1R failing to facilitate or D2R failing to repress neuronal excitability results in akinesia and rigidity: less movement. But how does D2R action repress indirect pathway MSNs?

Higley and Sabatini, 2010, show without doubt that one way is the following: D2R inhibits calcium entry in MSNs, particularly in their dendrites and spines, where most synaptic inputs arrive. Inhibition of calcium entry would then repress the excitability of D2R-expressing MSNs from the indirect pathway. Calcium entry into D2R-expressing indirect pathway MSNs is controlled by modulating two effectors: NMDAR and R-type Ca2+ channels—a synaptic (extrinsic) channel and a voltage-gated (intrinsic) calcium channel. In addition, inhibition of Ca2+ entry by D2R can be compensated for by A2AR-activation, which enhances Ca2+ entry. Experiments to delimit the signaling cascades involved were preliminary and need further work to be completely established.

Thus, in the same year (2010), the classical basal ganglia model of Albin et al. (1989) receives two strong empirical supports: first, normal behavior is due to the balance between direct and indirect basal ganglia pathways (Kravitz et al., 2010), and, second, indirect pathway excitability induced specifically by cortical afferents activation and subsequent Ca2+ inflow is reduced by D2R activation (Higley and Sabatini, 2010). Cortical terminals expressing channelrhodopsin on a single spine are stimulated by light and/or spine/dendritic synapses are stimulated directly by released caged glutamate. Thus, both pre- and post-synaptic components of the D2R-response are tackled.

Hyperdopaminergic syndromes (e.g., schizophrenia) monitored in rodents by evaluating hyperkinesia and hyperactivity, among other tests, are due to a dopamine excess acting on D1R-expressing MSNs (see discussion above). Conversely hypodopaminergic syndromes (e.g., parkinsonisms) are assumed to be due to lack of dopamine that impedes activation of D2R expressed in indirect pathway MSNs, thus abolishing the control over the excitability of MSNs, which therefore become hyperexcitable and begin to lose spines and synapses (A HREF="/pap/annotation.asp?powID=142792">Day et al., 2008). What does hyperexcitability mean here? According to Higley and Sabatini, Ca2+ inflow is not controlled and an excess of it occurs. Diminished D2R leads to D2R-expressing indirect pathway MSNs’ hyperexcitability, leading to akinesia and rigidity.

However, several antipsychotics act by inhibiting D2R, thus mimicking a lack of dopamine on these receptors, increasing indirect pathway activation, and thus overpowering direct pathway activation. Therefore, D2R inhibition has collateral actions, i.e., parkinsonism. Conversely, Kravitz et al. (2010) suggest that to alleviate parkinsonian motor deficits, one should overactivate D1R-expressing neurons to overcome hyperexcitable D2R-expressing neurons. It can be predicted, then, that a probable collateral action would be to cause the symptoms of schizophrenia.

Thus, one cannot focus on just one of these receptors or pathways (direct or indirect) to alleviate a given syndrome, as they represent a control system that dynamically reaches a balance between them in every situation. Seeing the problem this way poses a hard challenge for treating pathophysiology.

A complication for Higley and Sabatini's work is that at some stimulation protocols, NMDAR-antagonists increase, rather than decrease excitability. The same happens with R-type channels. The explanation is that Ca2+ entry activates SK-channels, and, in fact, Ca2+ entry may serve to decrease excitability. However, with other stimulation protocols, EPSPs and EPSCs go along (increase) with Ca2+ inflow; thus, Ca2+ entry increases excitability. The problem is: what really happens physiologically?

Kravitz et al., 2010 could not answer this question, because their dopamine experiments involved activity on the microcircuit, and thus their results were unpredictable or unexplainable. However, some additional work may get around this setback: first, supra-threshold cortical stimulation, that is, a stimulus capable of generating firing of action potentials as an up-state does, is a response that directly answers what happens with the firing of MSNs after Ca2+ entry in each pathway, direct or indirect. Recent experiments using supra-threshold cortical stimulation readily distinguished between direct and indirect pathway neurons, and, in fact, D2R-expressing indirect pathway neurons manage Ca2+ inflow differently from D1R-expressing direct pathway neurons (Flores-Barrera et al., 2010): D2R-expressing neurons exhibit synaptically driven auto-regenerative Ca2+ spikes which may reflect all the Ca2+ sources that Higley and Sabatini mention in their work. D1R-expressing neurons do not exhibit these spikes under the same conditions. In fact, inhibition plays almost opposite roles in indirect and direct pathway neurons due to this difference in Ca2+ management.

This is an important difference between MSNs from both pathways and supports the work by Higley and Sabatini. Experiments are needed to know what the precise Ca2+ source is to trigger the dendritic spikes, or alternatively, if a non-linear cooperation among all sources occurs. That will answer whether a VGCC antagonist may help in controlling D2R-expressing neuron hyperexcitability, thus diminishing the probability of collateral effects accompanying D2R-blockage. It also would be pharmacologically relevant to review (experimentally) the possible VGCC blocking properties of different antipsychotic classes, or, alternatively, to develop a drug with both properties, thus increasing synergy and requiring lower dose.

Previous work on single cells and on synaptic terminals have reported similar findings to those from Higley and Sabatini: D1R-activation increases direct pathway excitability in part through the enhancement of NMDAR and VGCC currents, and D2R-activation decreases indirect pathway excitability through the negative modulation (inhibition) of NMDAR and VGCC currents. The difference is that these previous studies showed these differences by recording at the somata (Cepeda and Levine, 1998; Hernandez-Lopez et al., 1997; 2000) or at the synaptic terminals (Guzman et al., 2003; Salgado et al., 2005; Tecuapetla et al., 2007). The elegance of Higley and Sabatini's work lies in that they demonstrate the same phenomena in spines and dendrites while recording their impact at the soma using a combination of highly sophisticated techniques—perhaps the best combination that can be used today. Taking all these studies together, we can conclude that D2R activation decreases or regulates Ca2+ entry at the soma, dendrites, spines, and synaptic terminals of D2R-expressing MSNs. That being said, it is still not clear what the impact of D2R-activation would be at the circuit level (see Kravitz et al., 2010), since a decrease in dendritic excitability and firing, plus a decrease in Ca2+ entry at the terminals, may lead to less GABA-release within the striatal circuitry and into the striatal target (GPe). The end result may be an increase in circuit excitability.

In relation to this paradox, Singer and his group posit that behind many brain disorders, including schizophrenia and Parkinson disease, there is a problem in circuit organization, such that low frequencies engage neurons in a dominant, hyperexcitable, and synchronized state that impedes normal circuit processing (Uhlhaas and Singer, 2006). This has been recently proven for Parkinson disease (Jaidar et al., 2010). A similar work has to be done in schizophrenia (Uhlhaas and Singer, 2010). Thus, the clue for global understanding may not be located on somata, dendrites, spines, or terminals, although this work makes the necessary bricks for making a solid cellular foundation, but in the circuit (Yuste, 2008).

Finally, Higley and Sabatini (2010) complete the ongoing work of finding a role for the different VGCCs found in neurons. At least for the MSNs, L(D)-type channels help to fix the threshold for firing, the frequency-intensity relationship, whereas N- and P/Q-type channels activate Ca2+-dependent potassium currents and the after-hyperpolarization, thus fixing the firing frequency and the firing pattern (more or less spike frequency adaptation) (Perez-Garci et al., 2003). P/Q-type channels are also in charge of synaptic release (Salgado et al., 2005). Thanks to Higley and Sabatini's work, we now know that R- and possibly T-type channels control arriving synaptic inputs at the dendrites, either by shunting them through Ca2+-dependent potassium currents or by boosting them. In addition, L(C)-type channels may generate auto-regenerative spikes. Perhaps this depends on input convergence and strength (Flores-Barrera et al., 2010). Now, each VGCC has at least one role in MSNs, and a great step forward for Ca2+ channels pharmacology would be to control a cellular function by modifying the Ca2+ channel that controls it.

References:

Kravitz AV, Freeze BS, Parker PR, Kay K, Thwin MT, Deisseroth K, Kreitzer AC. Regulation of parkinsonian motor behaviours by optogenetic control of basal ganglia circuitry. Nature. 2010 Jul 29;466(7306):622-6. Abstract

Albin RL, Young AB, Penney JB. The functional anatomy of basal ganglia disorders. Trends Neurosci. 1989 Oct;12(10):366-75. Abstract

Higley MJ, Sabatini BL. Competitive regulation of synaptic Ca2+ influx by D2 dopamine and A2A adenosine receptors. Nat Neurosci. 2010 Aug;13(8):958-66. Abstract

Day M, Wokosin D, Plotkin JL, Tian X, Surmeier DJ. Differential excitability and modulation of striatal medium spiny neuron dendrites. J Neurosci. 2008 Nov 5;28(45):11603-14. Abstract

Flores-Barrera E, Vizcarra-Chacón BJ, Tapia D, Bargas J, Galarraga E. Different corticostriatal integration in spiny projection neurons from direct and indirect pathways. Front Syst Neurosci. 2010 Jun 10;4:15. Abstract

Hernández-López S, Bargas J, Surmeier DJ, Reyes A, Galarraga E. D1 receptor activation enhances evoked discharge in neostriatal medium spiny neurons by modulating an L-type Ca2+ conductance. J Neurosci. 1997 May 1;17(9):3334-42. Abstract

Hernandez-Lopez S, Tkatch T, Perez-Garci E, Galarraga E, Bargas J, Hamm H, Surmeier DJ. D2 dopamine receptors in striatal medium spiny neurons reduce L-type Ca2+ currents and excitability via a novel PLC[beta]1-IP3-calcineurin-signaling cascade. J Neurosci. 2000 Dec 15;20(24):8987-95. Abstract

Cepeda C, Levine MS. Dopamine and N-methyl-D-aspartate receptor interactions in the neostriatum. Dev Neurosci. 1998;20(1):1-18. Abstract

Guzmán JN, Hernández A, Galarraga E, Tapia D, Laville A, Vergara R, Aceves J, Bargas J. Dopaminergic modulation of axon collaterals interconnecting spiny neurons of the rat striatum. J Neurosci. 2003 Oct 1;23(26):8931-40. Abstract

Salgado H, Tecuapetla F, Perez-Rosello T, Perez-Burgos A, Perez-Garci E, Galarraga E, Bargas J. A reconfiguration of CaV2 Ca2+ channel current and its dopaminergic D2 modulation in developing neostriatal neurons. J Neurophysiol. 2005 Dec;94(6):3771-87. Abstract

Tecuapetla F, Carrillo-Reid L, Bargas J, Galarraga E. Dopaminergic modulation of short-term synaptic plasticity at striatal inhibitory synapses. Proc Natl Acad Sci U S A. 2007 Jun 12;104(24):10258-63. Abstract

Uhlhaas PJ, Singer W. Neural synchrony in brain disorders: relevance for cognitive dysfunctions and pathophysiology. Neuron. 2006 Oct 5;52(1):155-68. Abstract

Jáidar O, Carrillo-Reid L, Hernández A, Drucker-Colín R, Bargas J, Hernández-Cruz A. Dynamics of the Parkinsonian striatal microcircuit: entrainment into a dominant network state. J Neurosci. 2010 Aug 25;30(34):11326-36. Abstract

Uhlhaas PJ, Singer W. Abnormal neural oscillations and synchrony in schizophrenia. Nat Rev Neurosci. 2010 Feb;11(2):100-13. Abstract

Yuste R. Circuit neuroscience: the road ahead. Front Neurosci. 2008 Jul;2(1):6-9. Abstract

Pérez-Garci E, Bargas J, Galarraga E. The role of Ca2+ channels in the repetitive firing of striatal projection neurons. Neuroreport. 2003 Jul 1;14(9):1253-6. Abstract

View all comments by Jose Bargas

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Related News: Commentary Brief: Optogenetics Links Interneurons and γ Oscillations

Comment by:  Guillermo Gonzalez-Burgos
Submitted 24 July 2009
Posted 24 July 2009

Blue light, yellow light, and the role of parvalbumin-positive neurons in the pathophysiology of schizophrenia
Parvalbumin (PV)-positive cells are a prominent subtype of GABA neuron that via perisomatic synapses may strongly inhibit pyramidal cell activity (however, see Szabadics et al., 2006). In schizophrenia, PV neurons have reduced levels of mRNA for PV and for GAD67, the 67 kilodalton form of the GABA-synthesizing enzyme glutamate decarboxylase. The functional consequences of the PV reduction in schizophrenia are poorly understood, but one possibility is that decreased PV partially compensates for a deficit in GABA release caused by the GAD67 reduction. PV is a slow Ca2+ buffer, and so decreasing PV in nerve terminals may facilitate GABA release during repetitive PV cell firing (for a review, see Gonzalez-Burgos and Lewis, 2008).

Why is PV cell-mediated inhibition significant to brain function? What deficits in cortical circuit function may be compensated for (at least partially) by a decrease of PV in schizophrenia? The answers to these questions depend on our knowledge of the functional role of PV neurons in cortical circuits. A leading hypothesis in this regard suggests that PV cells are essential for the production of synchronized oscillations in cortex, particularly in the γ frequency band (Bartos et al., 2007). γ oscillations are thought to be important for the transmission of information within and between neocortical areas (Salinas and Sejnowski, 2001). If so, then γ activity must be important for cognition, which is critically dependent on the flow of information across the neocortex (Singer, 1999; Fuster, 2004). Therefore, cognitive deficits (a key feature of schizophrenia) may result from the impairment of γ oscillations reported in the illness, which in turn could be a consequence of deficits in PV cell-mediated inhibition.

How is PV cell-mediated inhibition linked to the production of γ oscillations? PV neurons alone are not sufficient to produce network oscillations because, although PV cell membranes resonate at γ frequency (Pike et al., 2000), PV neurons actually lack intrinsic pacemaker activity. Therefore, whereas PV cells may be necessary to produce γ oscillations, synaptic interactions with other cell types in the cortical circuit must be necessary as well.

Studying the link between PV neuron activity and γ oscillations is complicated by the lack of tools available to selectively manipulate PV cell activity. Interestingly, two recent studies (Sohal et al., 2009; Cardin et al., 2009) employed novel “optogenetic” methods in mice, to further advance our understanding of how PV neurons are involved in γ oscillations.

Using viral vectors to drive cell type-specific expression of recombinant genes, Sohal and colleagues, as well as Cardin et al., produced expression of microbial opsins (which are light-sensitive ion channel proteins) selectively in specific populations of cortical neurons. Briefly described, modulation of neuronal activity using such optogenetic techniques works as follows: in cells expressing channelrhodopsin-2 (ChR2), illumination with blue light produces a depolarizing current that has excitatory effects (increases cell firing). On the other hand, cells expressing halorhodopsin (eNpHR) respond to yellow light with a hyperpolarizing current that has inhibitory effects (decreases cell firing). In this way, light of different wavelengths can be used to either inhibit or excite PV cells or pyramidal (PYR) neurons in the mouse cortex (in vivo or in vitro) in different experimental designs.

Sohal and colleagues first demonstrated that inhibiting the activity of eNpHR-expressing PV neurons with yellow light suppresses γ oscillations generated in vivo by rhythmic flashes of blue light applied to stimulate nearby ChR2-expressing PYR neurons. Furthermore, they show that non-rhythmic excitation of PYR cells produces non-rhythmic PYR cell firing. However, if the non-rhythmic PYR spikes are used to trigger feedback inhibition by PV neurons (driven by blue light flashes that stimulate ChR2-expressing PV cells), the addition of feedback inhibition induces a γ rhythm in PYR cell output. These results show that by means of recurrent interactions with pyramidal cells (consistent with the so-called PING models of γ rhythms; Whittington et al., 2000), PV cell activity is crucial for γ oscillations.

Finally, the experiments performed by Sohal et al. suggest that γ activity may selectively enhance the flow of information in cortical circuits. For example, their experiments demonstrate that the gain of the neuronal input-output relation (that is, the slope of the curve describing the transformation of inputs into outputs) is specifically enhanced when excitatory inputs onto a PYR cell are modulated rhythmically at γ frequency. Moreover, the amount of information flowing across synapses during interactions between PYR and PV neurons was estimated using concepts derived from information theory. The estimates from Sohal et al. showed that when network activity was driven by trains of blue flashes delivered at γ frequency, information transmission was markedly enhanced.

Using somewhat different genetic engineering approaches, Cardin and colleagues produced cell type-specific expression of ChR2 in PV cells or PYR neurons. Expression of ChR2 in PV cells of the mouse barrel (somatosensory) cortex allowed the activation of PV neurons with rhythmic flashes of light. Such manipulation produced rhythmic population activity (as detected recording local field potentials) more strongly when PV cells were driven at γ frequency compared with other frequencies. They report, in addition, some data showing that manipulation of PV cell activity has an impact on γ oscillations intrinsically generated by the cortical circuits, as opposed to γ rhythms induced by rhythmic stimuli applied by the investigators. For example, brief flashes of blue light applied to stimulate firing of ChR2-expressing PV neurons during spontaneous γ activity were able to reset the phase of the γ rhythm. Cardin et al. also demonstrate that activation of ChR2-expressing PV neurons can suppress the somatosensory response (to whisker stimulation) of nearby PYR cells. Then they go on to test an important functional role predicted for γ oscillations: that cells in a local network engaged in γ oscillations may respond differently to incoming inputs, depending on the timing of the incoming inputs relative to the phase of the γ cycle (Fries et al., 2007). In an elegant experiment, the investigators paired brief whisker stimulation with rhythmic flashes of blue light which, by stimulating ChR2-expressing PV cells, generate a γ rhythm locally. The crucial finding from this experiment is that the excitatory power of whisker stimulation was strongly dependent on the γ cycle phase at which whisker stimulation was delivered.

The data from these two recent studies briefly summarized above further consolidate the notion that PV-positive GABA neurons are key players in the mechanisms of γ synchrony in cortex. The data from these studies confirm that rhythmic PV neuron firing at γ frequency is sufficient to generate a γ rhythm in the population of postsynaptic neurons. This is not the same, however, as saying that PV neurons alone are sufficient to generate γ. Indeed, the data from these two studies point to the idea that PV neurons work in close interaction, via feedback loops, with nearby pyramidal cells. This makes sense, given that PV neurons are not intrinsic pacemakers. γ rhythms, therefore, seem to originate in complex network interactions that require the coordinated activity of pyramidal neurons, PV cells, and possibly other GABA neuron subtypes as well.

These two studies also highlight the similar importance of PV cell-dependent γ oscillations across different regions of cortex primarily involved in very different functions: Sohal and colleagues studied the role of PV neurons in frontal cortical areas, whereas Cardin et al. manipulated PV cell activity in the primary somatosensory cortex. The similarity of the findings regarding PV neuron function suggests that these neurons probably play a very similar role, at the microcircuit level, in these two very different cortical areas. Interestingly, deficits in GABA transmission, as assessed in postmortem brain studies, appear to be found in multiple areas of cortex simultaneously (Hashimoto et al., 2008). If this is indeed the case, then an impairment of γ oscillations may be present in most cortical areas. Thus, deficits in PV cell-dependent γ rhythms may explain the impairment of not only complex cognitive functions (for example, working memory), but also of more basic sensory processing which, as reviewed elsewhere (Javitt, 2009), is also impaired in schizophrenia. Finding a global deficit of GABA transmission and γ oscillations, as opposed to a deficit restricted to a single cortical area, increases the probability of success in developing pharmacological treatments.

A better understanding of the functional role of PV neurons in normal cortical circuits is crucial to developing better models that could explain how alterations of PV cells (and of other GABA neurons subtypes) originate in schizophrenia. As highlighted elsewhere (Lewis and Gonzalez-Burgos, 2008), any given alteration observed in schizophrenia may represent 1) cause, an upstream factor related to the disease pathogenesis; 2) consequence, a deleterious effect of a cause; 3) compensation, a response to either cause or consequence that helps restore homeostasis; or 4) confound, a product of factors frequently associated with, but not a part of, the disease process, or an artifact of the approach used to obtain the measure of interest. A major challenge for schizophrenia research is therefore determining to which of the four “C” categories each alteration belongs (Lewis and Gonzalez-Burgos, 2008). Certainly, information from basic neuroscience research studies such as those of Sohal et al. and Cardin et al. and many other past and future studies is extremely helpful in achieving this goal.

References:

Szabadics J, Varga C, Molnar G, Olah S, Barzo P, Tamas G: Excitatory effect of GABAergic axo-axonic cells in cortical microcircuits. Science 311:233-235, 2006. Abstract

Gonzalez-Burgos G, Lewis DA: GABA Neurons and the Mechanisms of Network Oscillations: Implications for Understanding Cortical Dysfunction in Schizophrenia. Schizophr Bull 34:944-961, 2008. Abstract

Bartos M, Vida I, Jonas P: Synaptic mechanisms of synchronized gamma oscillations in inhibitory interneuron networks. Nat Rev Neurosci 8:45-56, 2007.Abstract

Salinas E, Sejnowski TJ: Correlated neuronal activity and the flow of neural information. Nat Rev Neurosci 2:539-550, 2001. Abstract

Singer W: Neuronal synchrony: a versatile code for the definition of relations? Neuron 24:111-25, 1999. Abstract

Fuster JM: Upper processing stages of the perception-action cycle. Trends Cogn Sci 8:143-145, 2004. Abstract

Pike FG, Goddard RS, Suckling JM, Ganter P, Kasthuri N, Paulsen O: Distinct frequency preferences of different types of rat hippocampal neurones in response to oscillatory input currents. J Physiol 529 Pt 1:205-213, 2000. Abstract

Sohal VS, Zhang F, Yizhar O, Deisseroth K: Parvalbumin neurons and gamma rhythms enhance cortical circuit performance. Nature 2009 Jun 4;459(7247):698-702. Abstract

Cardin JA, Carlen M, Meletis K, Knoblich U, Zhang F, Deisseroth K, Tsai LH, Moore CI: Driving fast-spiking cells induces gamma rhythm and controls sensory responses. Nature 2009 Jun 4;459(7247):663-7. Abstract

Whittington MA, Traub RD, Kopell N, Ermentrout B, Buhl EH: Inhibition-based rhythms: experimental and mathematical observations on network dynamics. Int J Psychophysiol 38:315-336, 2000. Abstract

Fries P, Nikolic D, Singer W: The gamma cycle. Trends Neurosci 30:309-316, 2007. Abstract

Hashimoto T, Bazmi HH, Mirnics K, Wu Q, Sampson AR, Lewis DA: Conserved Regional Patterns of GABA-Related Transcript Expression in the Neocortex of Subjects With Schizophrenia. American Journal of Psychiatry 162:479-489, 2008. Abstract

Javitt DC: When doors of perception close: bottom-up models of disrupted cognition in schizophrenia. Annu Rev Clin Psychol 5:249-275, 2009. Abstract

Lewis DA, Gonzalez-Burgos G: Neuroplasticity of neocortical circuits in schizophrenia. Neuropsychopharmacology 33:141-165, 2008. Abstract

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