New Insights on Cortical Neuron Plasticity

All procedures and use of animals were approved by the University of Michigan Institutional Animal Care and Use Committee. Mouse lines were obtained from Jackson Laboratories unless specified. The following mouse lines were used for whole cell recordings: Ai14, Ai32, C57BL/6J, Cux2-Cre, Grp-Cre, Htr2a-floxed (Htr2afl), Kj319. Nex-Cre, Pvalb-Ai14, Pvalb-Cre, Scnn1a-Cre, Syt6-Cre. All mice used in the experiments were bred on a C57BL6 background (Charles River). A combined total of 321 neurons from 84 mice of both sexes between the ages of postnatal days 30–306 were used.

Creation and validation of Htr2a conditional knockout mice

With the University of Michigan Transgenic Animal Model Core, we utilized the CRISPR-Cas9 system in C57BL/6 mouse embryos to introduce two LoxP sites flanking the Htr2a Exon 1 protein-coding region (Htr2afl). Single-stranded RNA guides (Supplementary Table 1) were complexed with Integrated Data Technologies HiFi Cas9 protein (IDT # 1081060) before co-injection with donor DNA into embryos. HiFi Cas9 protein is designed for high specificity of on-target activity [62]. To validate that the LoxP insertions were successful, we performed PCR genotyping using the primers specified in Supplementary Table 1. G0 founders were bred with wild-type mates to create heterozygous G1 mice, which we bred in-house to homozygosity (Htr2afl/fl) before use in experiments.

RNAScope was used to confirm presence of Htr2a mRNA in prefrontal cortex (PFC) and anterior dorsal (AD) thalamus. RNAScope was also used to validate loss of Htr2a mRNA after viral transfection of Cre. We used three Htr2afl/fl mice injected with AAV2-Cre-eGFP into PFC (P93-162) as described in the Surgical Procedures. The virus was allowed to incubate for at least 4 weeks before the start of the RNAScope assay. Two of the three animals were deeply anesthetized with isoflurane, perfused, and the brain dissected into 10% neutral buffered formalin (Sigma #HT501128) for overnight fixation. Next day the brain was moved to 70% ethanol and used for paraffin embedding and subsequent RNAScope. One animal was deeply anesthetized with Isoflurane, decapitated, and the brain was dissected and immediately snap-frozen on dry ice and kept at −80 °C until the start of the experiment (7 days). Then, the brain was sliced on a cryostat (Leica 3050S) at 14 μm and mounted onto charged slides (Fisher Scientific #12-550-15). Mounted slices were kept overnight at −80 °C degrees and used next day for RNAScope.

Immediately prior to starting the assay, frozen slides were placed in 10% neutral buffered formalin for 1 h fixation at 4 °C, then dehydrated in series ethanol steps (50, 70, and 100%). Afterwards, a barrier was drawn around each slice using a hydrophobic pen (ACD # 310018), and all the subsequent reagents used were part of the RNAScope® Multiplex Fluorescent Reagent Kit v2 (ACD #323100) unless indicated otherwise. Endogenous peroxidase activity was then blocked with hydrogen peroxide solution, and the tissue permeabilized with protease IV.

After pretreatment, a custom Htr2a probe was prepared (ACD). The custom Htr2a probe was designed to target the floxed sequence of the Htr2a gene in the Htr2afl/fl mouse line. The probe was applied to slices, allowed to hybridize for 2 h at 40 °C and was then washed off using the wash buffer. The probe were then amplified using a series of three amplification steps, and fluorescent signal was developed using TSA Vivid 650 (ACD #323273). The signal development round was carried out at 40° C and consisted of the channel-specific HRP incubation, fluorescent dye application, and HRP blocking, with wash buffer washes after each step. Finally, the slices were mounted with FluoromountG with DAPI (Southern Biotech #0100-20), cover slipped, and set overnight at 4 °C. Confocal imaging was then carried out.

25CN-NBOH was purchased from Tocris Bioscience and was administered to mice via intraperitoneal (IP) injection (2, 10, or 20 mg/kg, specified throughout text), dissolved in sterile saline, brought up to a volume equaling no more than 1% of the mouse’s total bodyweight. Brief sonication was used to aid solubility. Mice were moved to single housing before injection and recorded 24–72 h post-injection. For acute pharmacology recordings, 25CN-NBOH (final concentration = 10 µM) was diluted into artificial cerebral spinal fluid (ACSF) from a stock concentration dissolved in sterile water.

Surgical anesthesia was induced via vaporized isoflurane inhalation at 5% and then maintained at 1–3% isoflurane. Upon induction, atropine was injected subcutaneously at 0.05 mg/kg. A Physitemp (Clifton, New Jersey, USA) controller monitored and maintained body temperature at 37 °C. Ophthalmic ointment was placed on the eyes. The incision site was prepared using a wash of Nolvasan (1:40) followed by isopropyl alcohol and then a subcutaneous injection of lidocaine (1%). The skull was cleaned using Hydrogen Peroxide, then leveled using bregma and lambda. Using a digital stereotaxic coordinate system, the following injection target sites were identified: right anterodorsal (AD) thalamic nucleus for ChR2 experiments (AP = −0.6 mm, ML = ± 0.78 mm, DV = −2.5, –3.25 mm), and both AD thalamic nuclei for Cre-GFP injection. Craniectomies were performed at the target sites, and then dura at the target coordinates was removed. Micropipettes were lowered under stereotaxic guidance into the target injection site containing the ChR2 viral construct (AAV2-EF1a-DIO-hChR2(h134R)-eYFP or Cre/GFP viral construct (AAV2-hSyn-GFP-Cre), obtained from UNC Gene Therapy Vector Core. Injections of 2.0 µL total virus volume were administered via a picospritzer (0.05–0.07 µL/min). After injection, there was a 10-min period before removing the micropipette from the brain. Enrofloxacin was administered (8.0 mg/kg) after injections. Craniectomies were sealed with bone wax, the skin incision was closed with VetBond, and with antibiotic ointment placed under skin edges. Isoflurane was tapered down prior to removal, after which carprofen was administered (5 mg/kg). Mice were kept warm through an artificial heat source during the recovery period. Mice then recovered for 3–6 weeks post-injection before being used in experiments.

Brains were dissected in ice-cold sucrose-substituted ACSF, saturated with 95% O2 and 5% CO2 and containing the following (in mM): 3 KCl, 1.25 NaH2PO4, 26 NaHCO3, 10 dextrose, 234 sucrose, 0.2 CaCl2, 4 MgSO4. 300 μm coronal slices were cut using a Leica Microsystems 1200VT vibratome and placed in a high-magnesium ACSF solution at 32 °C for 30 min and subsequently rested at room temperature for at least 30 more minutes before being recorded. During experiments, slices were submerged in a recording chamber with physiological temperature (32 °C) ACSF (126 mM NaCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 3 mM KCl, 10 mM dextrose, 1.20 mM CaCl2, and 1 mM MgSO4) perfused at 3 mL/min. Slices containing retrosplenial cortex (RSC) were obtained from anteroposterior (AP) distance to bregma −1.0 to −3.7 mm; slices containing anterior cingulate cortex (ACC) were obtained from AP 2.1 to −0.5 mm; slices containing anterodorsal thalamus (AD) were obtained from AP −0.4 to −0.9 mm.

Whole-cell electrophysiology and quality control

Neurons were visualized on an Olympus BX51WI microscope, Olympus 60× water immersion lens, and Andor Neo sCMOS camera (Oxford Instruments, Abingdon, Oxfordshire, UK). Patch electrodes were pulled from borosilicate glass (Sutter Instruments; diameter 1.5 × 0.86 mm) to a tip resistance between 4–6 MΩ. The internal solution was potassium gluconate-based and contained (in mM): 130 K-gluconate, 0.6 EGTA, 10 HEPES, 2 MgATP, 0.3 Na2GTP, 6 KCl, NaCl and 0.5% biocytin (calculated ECl = −68 mV; pH = 7.25; osmolarity = 290 mOsm). Pipette capacitance compensation and bridge balance were applied; recordings were not corrected post-hoc for liquid junction potential. In ACC, only L5 neurons with regular spiking firing patterns that did not fire rebound spikes after hyperpolarizing current injection were used in this study. In RSG, LR cells in L2/3 were identified by characteristic firing patterns as previously described [48, 49, 53]. Cells were excluded if baseline resting membrane potential was more depolarized than −55 mV (or −45 mV for AD thalamus recordings), if input resistance decreased >25% before acute pharmacology experiments, if uncompensated series resistance (Rs) was >35 MΩ, or if Δ Rs >25% during acute pharmacology experiments. All whole-cell recordings were conducted with MultiClamp 700B amplifier and digitized at 20 kHz with Digidata 1550B (Molecular Devices) for collection on a computer equipped with pClamp 10.7 software (Molecular Devices). Patch electrodes additionally contained biocytin (0.5%), which enabled post-hoc imaging (see methods below) to determine somatic position and confirm cellular morphology. In ACC, we considered L5 neurons to have cell bodies 125 to 400 microns from the L1/2 border. In RSG, L2/3 was identified visually by the densely packed granular cell layer.

Cell filling and imaging

Internal solutions for all recorded neurons contained biocytin to enable post-hoc morphological analysis [29, 48, 49, 63, 64]. Cells were recorded for at least 15 min to allow diffusion of biocytin throughout the neuron. Upon completion of recording, the pipette was carefully retracted to enable membrane resealing and the slice was immediately transferred to 4% PFA for overnight fixation. After fixation, slices were washed in phosphate buffer solution (PBS) three times (ten minutes) and incubated in PBS containing 0.4% triton, fluorescent nissl (NeuroTrace 435/455), and 1:1000 streptavidin conjugated Alexa Fluor 647 at 4 °C (48 h). Afterwards, slices were washed in PBS, mounted with FluoromountG and allowed to rest for at least 24 h before imaging. Imaging was conducted with a Zeiss Axio Image M2 microscope equipped with LSM 700 confocal system using a 63× oil objective lens as z-stacks with z-step of 0.4 µm. Neurons were then reconstructed from these files using NeuTube 1.0 software.

Spine classification and density analysis

Spines were analyzed by an experimenter blinded to experimental conditions. For each neuron imaged, a representative main apical dendrite as well as apical tuft dendrites we imaged using a Zeiss Airyscan system. Dendrite lengths were measured in FIJI ImageJ. Spine counts were tracked using the Cell Counter plugin in FIJI. Cells that filled uniformly and had no major cut off branches were selected for further spine analysis. Z-stacks of the dendrites were obtained using a Zeiss Airyscan confocal microscope system. The z-stacks were processed in ImageJ, and the spines manually identified and counted with the “cell counter” plugin based on visual inspection. Spines are defined by visibly clear protrusions on dendrites. Up to 5 apical dendrites and up to 5 basal dendrites were selected for each neuron, and each dendrite was independently analyzed. To enable a reliable spine density measurement, branches shorter than 40 microns were not included. Note that LR neurons have fewer, thinner, and smaller dendritic branches than RS cells. The spine density for each dendrite was calculated as the number of spines on the segment divided by the length of the dendrite. For spine reconstructions, max projections of Z-stacks were obtained from the high resolution 3-D images for each group, and 5 um scale bars are added to the images with the ‘Scale Bar’ tool from ImageJ. One representative dendrite was then selected from each group and manually traced with Procreate.

Analysis of spontaneous synaptic activity

Voltage-clamp recordings were conducted at a holding potential of −70 mV. sEPSC recordings were taken in 30-second sweeps with a brief (250 ms) hyperpolarizing test pulse (−5 mV) at the start to monitor Rs and Rin throughout the experiment. Spontaneous excitatory post-synaptic currents (sEPSCs) were analyzed by an experimenter blinded to experimental conditions using Easy Electrophysiology (version 2.4.0) in 29-second sweeps (the first second of each sweep contained the test pulse and was thus discarded). Putative events were identified with threshold-based detection (negative peak direction, 5 ms local maximum period, 30 ms decay search period, 8 pA threshold, 10 ms search period, 1-ms averaged baseline, curved baseline and threshold). Events were manually inspected, and noise events were rejected to eliminate false positive events. Frequency was determined for each sweep by diving the total number of events per sweep by 29 s, amplitude was computed by averaging all events for each sweep, half-width and decay time constant were determined from fitting an exponential to the averaged event for each sweep. During acute pharmacology experiments, sEPSCs are recorded for two consecutive sweeps in standard ACSF, immediately before switching to the drug-containing ACSF and recording sEPSCs for 20 more sweeps. In some cases, optogenetic stimulation (described below) was additionally used during acute pharmacology experiments. Stimulation occurred after the test pulse, and thus the first 2 s of every sweep were discarded for sEPSC analysis.

Analysis of optogenetic-evoked synaptic activity

Optogenetic brain slice experiments were conducted on the same rig set-up using a 5500 K white light-emitting diode (LED; Mightex; maximum power of 14.47 mW measured at the slice focal plane). Synaptic responses to optical stimulation of the ChR2-expressing thalamic axons were measured from postsynaptic retrosplenial neurons recorded under whole-cell current-clamp or voltage-clamp conditions while optically stimulating L3. Synaptic responses were obtained from 10 Hz stimulation (10 pulses, 1 ms pulse-width, 4–5 trials). During voltage-clamp pharmacology experiments, after establishing a baseline response (1 ms pulse-width, 30 s sweeps), 25CN-NBOH was bath-applied for 10 min and RMP and evoked EPSC amplitude continued to be recorded under identical stimulation parameters. After the 10-min perfusion, the 10 Hz voltage clamp synaptic responses were again recorded (10 pulses, 1 ms pulse-width, 5 trials). The amplitude of the first pulse (pA), and the area (pA*ms) of each pulse (50 ms) were analyzed. To determine short-term synaptic transmission dynamics, each pulse in the 10 Hz train was normalized to the first pulse in both baseline and drug conditions.

Analysis of intrinsic physiological properties