Emergence of 5-HT5A signaling in parvalbumin neurons mediates delayed antidepressant action.

The behavioral response to antidepressants is closely associated with physiological changes in the function of neurons in the hippocampal dentate gyrus (DG). Parvalbumin interneurons are a major class of GABAergic neurons, essential for DG function, and are involved in the pathophysiology of several neuropsychiatric disorders. However, little is known about the role(s) of these neurons in major depressive disorder or in mediating the delayed behavioral response to antidepressants. Here we show, in mice, that hippocampal parvalbumin interneurons express functionally silent serotonin 5A receptors, which translocate to the cell membrane and become active upon chronic, but not acute, treatment with a selective serotonin reuptake inhibitor (SSRI). Activation of these serotonergic receptors in these neurons initiates a signaling cascade through which Gi-protein reduces cAMP levels and attenuates protein kinase A and protein phosphatase 2A activities. This results in increased phosphorylation and inhibition of Kv3.1β channels, and thereby reduces the firing of the parvalbumin neurons. Through the loss of this signaling pathway in these neurons, conditional deletion of the serotonin 5A receptor leads to the loss of the physiological and behavioral responses to chronic antidepressants.

0.5 CaCl 2 , 5 L-Ascorbic Acid, 3 Sodium Pyruvate, 2 Thiourea (pH was around 7.4, with osmolarity of 295-305 mOsm). After cutting, slices were left to recover for 15 minutes in the same cutting solution at 35 °C and for 1 h at room temperature (RT) in recording solution (see below). Whole-cell patch-clamp recordings were performed with a Multiclamp 700B/Digidata1550A system (Molecular Devices, Sunnyvale CA, USA). EGFP-positive PV neurons were selected for recording based on the expression of the fluorescent marker using an upright Olympus BX51WI microscope equipped with the appropriate filters (Olympus, Japan) and a SPECTRA X LED light engine (Lumencor, OR, USA). The extracellular solution used for all recordings contained (in mM): 125 NaCl, 25 NaHCO 3 , 2.5 KCl, 1.25 NaH 2 PO 4 , 2 CaCl 2 , 1 MgCl 2 and 25 glucose (bubbled with 95% O 2 and 5% CO 2 ). The slice was placed in a recording chamber (RC-27L, Warner Instruments, USA) and constantly perfused with oxygenated aCSF at 24 °C (TC-324B, Warner Instruments, USA) at a rate of 1.5-2.0 ml/min. For measuring 5-HT-induced responses, whole-cell current-clamp recordings were obtained from PV neurons using recording pipettes (King Precision Glass, Inc, Glass type 8250) pulled in a horizontal pipette puller (Narishige) to a resistance of 3-4 MΩ. The intracellular solution contained (in mM): 126 K-gluconate, 4 NaCl, 1 MgSO 4 , 0.02 CaCl 2 , 0.1 BAPTA, 15 glucose, 5 HEPES, 3 ATP, 0.1 GTP (pH 7.3). For measuring the membrane potential 30 seconds of recording were binned into 0.5 ms bins and fitted with a Gaussian. 5-HT (1-10-30-100 µM) and SB-669,551 (10 μM) were applied in the recording chamber using an automatic valve MPS-2 multichannel perfusion system (World Precision Instruments, USA). Tetradotoxin (TTX,0.3 μM) was added in the bath to prevent indirect responses from other neurons in the slices.
For measuring the action potential firing we used whole-cell current-clamp recordings from PV neurons. The intracellular solution contained (in mM): 126 K-gluconate, 4 NaCl, 1 MgSO 4 , 0.02 CaCl 2 , 0.1 BAPTA, 15 glucose, 5 HEPES, 3 ATP, 0.1 GTP (pH 7.3). Small currents were injected to the cells to bring the membrane potential at -70 mV. Consecutive current steps of 100 pA were injected to induce depolarization. The frequency of action potentials was measured using the first two action potentials evoked by the respective injected current. Action potential properties were measured from the first action potential to avoid any confounding effects of adaptation.
For measuring the Kv we used whole-cell voltage-clamp recordings from PV neurons. The intracellular solution contained (in mM): 126 K-gluconate, 4 NaCl, 1 MgSO 4 , 0.02 CaCl 2 , 0.1 BAPTA, 15 glucose, 5 HEPES, 3 ATP, 0.1 GTP (pH 7.3). Voltage steps of 10 mv (from −70 mV to 50 mV; 1s, every 10 s) were used to determine current-voltage (I-V) relationships. A P/4 protocol was used to eliminate leak currents. The amplitude of the current was measured on the steady-state part of the response. Tetradotoxin (TTX, 0.3 μM) was added in the bath to block Na + currents.
For measuring the GIRK we used whole-cell voltage-clamp recordings from PV neurons. The intracellular solution contained (in mM): 126 K-gluconate, 4 NaCl, 1 MgSO 4 , 0.02 CaCl 2 , 0.1 BAPTA, 15 glucose, 5 HEPES, 3 ATP, 0.1 GTP (pH 7.3).Voltage steps of 10 mv (from −120 mV to -70 mV; 1s, every 10 s) were used to determine current-voltage (I-V) relationships. A P/4 protocol was used to eliminate leak currents. The amplitude of the current was measured on the steady-state part of the response. Tetradotoxin (TTX, 0.3 μM) was added in the bath to block Na + currents.
For measuring the Gi-DREADD and Gs-DREADD effect on the membrane potential, mCherrypositive PV neurons were selected for recording based on the expression of the fluorescent marker. CNO (2 μM) was added to the bath and the membrane potential was measured as described above.
Data were acquired at a sampling frequency of 100 kHz, filtered at 1 kHz and analyzed offline using pClamp10 software (Molecular Devices, Sunnyvale, CA, USA). All electrophysiological data are expressed as means ± SEM. Statistical analysis was performed using the Student's ttest, One Way Analysis of Variance with Bonferroni post hoc comparison unless stated otherwise, with the help of GraphPad Prism 5. In all experiments, P < 0.05 was considered significant.