Neuronal signature of an antipsychotic response

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Antipsychotic drugs (APDs) are widely used to treat psychosis in schizophrenia 1,2 and symptoms in other neuropsychiatric conditions 3 . Blocking dopamine signaling onto 60-80% of striatal D2 receptors (D2r) is thought to underlie their therapeutic efficacy 4,5 . However, studies showing symptom improvement at a broader range of D2r blockade (i.e. 16-95%) [6][7][8][9][10] suggest that this mechanism is poorly predictive of antipsychotic response. Assessment of APD function at a circuit level has proven more predictive of behavioral efficacy in humans [11][12][13] and rodents 14 . In human patients, the effects of APDs on striatal circuitry are assessed from fMRI BOLD signal, which is inhibited by APDs 11,12 . This clinical response is at odds with expectations, given the physiology of dopamine receptor expressing striatal cells that intracellularly couple to either G i/o (D2r) or G s/o (D1 receptors, D1r), thereby leading to cell inhibition and stimulation, respectively 15,16 . Since APDs are antagonists or inverse agonists of D2r 17 , but largely spare D1r, the hyperdopaminergic signaling thought to underlie psychosis shifts to D1r in the presence of APDs, since D2rs are occupied, and the net striatal response is expected to be excitation 15 . Thus, despite the broad clinical application of APDs, the neurobiology underlying their psychomotor effects is unclear and difficult to explain based on mere D2r blockade.
To clarify these mechanisms, we used single cell in vivo calcium (Ca 2+ ) imaging to analyze activity of D1rand D2r-expressing neurons in freely moving mice at baseline and in response to an acute intraperitoneal injection of the typical APD haloperidol (HAL 0.5mg/kg, Fig. 1A), a widely prescribed APD with high affinity for D2r 18 . Striatal cells respond to HAL within minutes 19 , contributing to changes in brain structure 20 and symptoms 21 within hours. We determined the earliest psychomotor effects of HAL in relationship with D1r and D2r neuronal responses in the nucleus accumbens core (NAcc), a prominent striatal structure involved in spontaneous locomotion in animals 22,23 and in psychosis and antipsychotic responses in humans [11][12][13]24 .
Because medium spiny neurons (MSNs) are the most prevalent neuronal type in the striatum (~95%) in humans and animals 25 for simplicity we will refer to labeled cells as MSNs throughout the text. We used D1-and D2-cre transgenic mice 26 to obtain selective expression of the Ca 2+ sensing fluorophore GCaMP6f in D1-or D2-MSNs (Fig. 1B), recorded with gradient refractive index lenses connected to a head mounted miniature microscope ( Fig.1C and methods). We measured Ca 2+ events from a total of 568 MSNs (246 D1-MSNs and 322 D2-MSNs from 8 mice each) over 45 min (15 min baseline and 30 min after HAL or saline injection ( Fig. 1D-E) while animals moved freely in an open field to which they were previously habituated.
At baseline D1-MSNs were more active than D2-MSNs ( Fig. 2A). In animals that received saline, D1-but not D2-MSN activity correlated positively with locomotion (Fig. S1). An acute injection of HAL did not reduce spontaneous locomotion relative to saline-injected animals ( Fig 2B) and did not induce catalepsy, but moderately impacted reward reactivity (Fig S2-3 MSN firing rate is not constant, but alternates between periods of relative silence and episodes of moderate or high firing 27,28 . For this reason, the depression of D2-MSN activity after HAL could result from complete abolition of Ca 2+ events (i.e. no firing activity) or instead could result from an activity switch from high-tolow firing. We analyzed the cumulative frequency distributions of Ca 2+ events at baseline and after HAL treatment and found that HAL did not completely suppress Ca 2+ events, but instead decreased the number of D2-and D1-MSNs firing at high frequency ( Fig. 2G-H). By subdividing MSNs into quartiles according to frequency of Ca 2+ events at baseline, we found that HAL decreased the proportion of D2-MSNs exhibiting high and moderate Ca 2+ spike frequency gradually over the 30 min recording session, whereas the proportion of D2-MSNs with low or no Ca 2+ events were ultimately ~80% of all D2-MSNs (Fig. 2I).
No changes in firing frequency were observed in D1-MSNs after HAL during the first 20-min of the recording session, but a significant shift was observed during the last 10 min of recording, where the proportion of cells firing at high frequency was reduced compared to baseline (Fig. 2J). Since NAcc MSN firing is the result of a summation of excitatory and inhibitory inputs 29,30 , we compared the increased/decreased ratio of MSN activity for each animal based on the number of neurons firing above or below the median at baseline to estimate the net effect of HAL on MSN activity. Confirming the results in  S5). Because endogenous D2r, but not D1r on MSNs can couple to GIRK2 channels, GIRK2 functions as a sensor providing a rapid, direct readout of IPSC-mediated synaptic D2r activation (D2r-IPSC) 35,36 . As expected, synaptic dopamine stimulation evoked D2r-IPSCs in NAcc D2-MSNs in control animals and HAL reduced it five-fold, (Fig. 3A). The lag to IPSC onset and decay increased after HAL treatment indicating a delayed NAcc D2r-IPSCs (Fig. 3B) and reduced rate of dopamine clearance, respectively (Fig.   3C). Importantly, we estimated that ~21-45% of D2r were spared by HAL depending on incoming dopaminergic transmission, with a more robust D2r-IPSC reduction following increased dopamine transmission. Indeed, HAL suppressed only 44-66% of D2r-IPSC at low intensity stimulation and 76-81% at higher stimulation (Fig. 3D).
Together, these data indicate that HAL effectively reduced post-synaptic dopamine signaling onto NAcc D2-MSNs, but also that endogenous dopamine could stimulate a pool of spared NAcc D2r even in the presence of HAL. The reduced D2r-IPSC decay indicates reduced dopamine clearance, which could prolong dopamine transmission onto both MSN subtypes (Fig. 2C-D). While we recapitulated previous reports on partial receptor occupancy 14,34 and dopamine uptake blockade 14 30 . To test this hypothesis, we conducted ex vivo whole-cell electrophysiological recordings from NAcc D2-MSNs in control and HAL treated mice. We first determined whether glutamate release probability was altered by HAL using the paired-pulse ratio (PPR) of evoked excitatory postsynaptic currents (EPSCs). PPR was significantly reduced on D2-MSNs of HAL treated mice (Fig. 4A), excluding that reduced excitatory transmission onto D2-MSNs contributed to D2-MSN suppression after HAL. Next, we evaluated putative post-synaptic alterations by measuring the ratio of α -amino-3-hydroxy-5-methyl-4isoxazolepropionic acid receptors (AMPArs) and N-methyl-D-aspartate receptors (NMDArs).

Pharmacological isolation of AMPAr and NMDAr currents revealed an enhanced AMPAr/NMDAr in D2-
MSNs in HAL treated mice (Fig. 4B). While increased AMPAr/NMDAr may broadly indicate enhanced synaptic strength (i.e. long-term potentiation, LTP) in D2-MSNs, an imbalanced AMPAr/NMDAr could also result from decreased NMDAr rather than increased AMPAr currents, which is likely to reduce synaptic strength.
To determine whether a systemic HAL injection modified NMDAr currents, we examined the currentvoltage (I/V) relationship of pharmacologically isolated NMDAr currents in D2-MSNs. NMDAr I/V curves in D2-MSNs were significantly decreased in HAL treated mice at -20 mV compare to control (Fig. 4C), suggesting that NMDArs undergo gross changes in voltage dependence. Moreover, the NMDAr current at +40 mV revealed faster decay kinetics in mice treated with HAL (Fig. 4D), independently of differences in membrane properties, as membrane capacitance and input resistance did not differ between treatment groups (Table S2). To confirm that HAL facilitated presynaptic, but depressed post-synaptic excitatory transmission we assessed spontaneous EPSC (sEPSC) frequency and amplitude and found that D2-MSNs in HAL treated mice showed a significant increase in sEPSC frequency, but not amplitude (Fig. 4E).
HAL is a potent inhibitor of the sigma receptor 40 , which is known to regulate pre-and post-synaptic glutamate transmission 41,42 . To test if HAL altered NMDA receptor function by blocking the sigma receptor we bath applied the sigma receptor agonist siramisine and found that while siramisine significantly decreased NMDA current in D2-MSNs of control animals, this effect was antagonized in HAL-treated animal (Fig.4F). Together, these findings show that HAL alters NMDA excitatory transmission likely through blockade of sigma receptor function, likely leading to alterations in synaptic plasticity. Because synaptic plasticity relies on Ca 2+ influx through NMDAr 43 we expected that the impact of HAL on NMDAr function would alter synaptic plasticity within the striatal network. To determine whether decreased NMDAr function after HAL altered the signature of synaptic plasticity, we measured the amplitude of field EPSPs after the application of high-frequency stimulation (HFS) of glutamatergic afferent fibers in the NAcc. In normal conditions the application of HFS enables Ca 2+ entry through post-synaptic NMDAr and triggers NMDAr-dependent LTP 43 . Accordingly, the amplitudes of field EPSPs were significantly increased from control mice (Fig. 4E) after HFS. To examine whether this LTP was mediated by NMDAR activation, field EPSPs were recorded in the presence of the selective NMDAR antagonist D-AP5, which inhibited synaptic strength expression (Fig. 4E) and confirmed the NMDAR-dependency of LTP. Since HAL shortened NMDAr decay kinetics, thereby reducing the amount of Ca 2+ entry through NMDAr, we predicted that LTP magnitude in the NAcc would be reduced by HAL. Indeed, as shown in (Fig. 4F-G), application of HFS decreased field EPSP amplitude below baseline after HAL, demonstrating that the same protocol that potentiated synaptic transmission in control animals, instead depressed synaptic transmission in mice receiving HAL.
We reveal for the first time rapid neuronal responses of an antipsychotic in relationship to psychomotor output, which is largely independent of D2 receptor blockade. Importantly, locomotion was correlated with NAcc D2-MSNs Ca 2+ activity after HAL. The suppression of Ca 2+ events in NAcc D2-MSNs were underlined by shortened NMDAr offset, by antagonism of the sigma receptor and impaired LTP.
Paradoxically, these physiological effects emerged despite HAL efficiently blocking dopamine transmission onto ~73% of D2r, minimizing the role of D2r blockade in an antipsychotic response. Because HAL reduced D2-IPSCs without suppressing them completely, our findings suggest that spared D2r might also have mediated the reduced Ca 2+ signaling and LTP expression. These rapid changes in D2-MSN functional plasticity rather than D2r blockade are likely to contribute to rapid symptom improvements 21,44 .
Finally, the depression of MSN responses illuminates the neurobiology underlying reduced striatal BOLD signals observed in human studies coincident with antipsychotic efficacy [11][12][13] . Here we extend these fMRI studies by showing divergent D1-and D2-MSN responses to an APD, a cell-type signature that cannot be captured by fMRI. Importantly, because in our study a main mechanism of action of HAL was to induce synaptic meta-plasticity by blocking LTP induction, our results shed light on why antipsychotic responses can endure even after treatment discontinuation in humans 45 and animals (Fig. S3).