Protocol for quantifying pyramidal neuron hyperexcitability in a mouse model of neurodevelopmental encephalopathy

Abstract

Here, we present a protocol for quantifying pyramidal neuron hyperexcitability in a mouse model of STXBP1 neurodevelopmental encephalopathy (Stxbp1^hap). We describe steps for preparing brain slices, positioning electrodes, and performing an excitability test to investigate microcircuit failures. This protocol is based on recording layer 2/3 cortical pyramidal neurons in response to stimulation of two independent sets of excitatory axons that recruit feedforward inhibition microcircuits. For complete details on the use and execution of this protocol, please refer to Dos Santos et al.¹.

Keywords: Genetics; Health Sciences; Neuroscience.

Graphical abstract

Figure 1

Positioning the stimulation electrodes on L4 from the somatosensory cortex (A) Schema of a sagittal section from the mouse brain. Cx: Cortex, Hp: Hippocampus, Mo: Motor, Ss: Somatosensory, St: Striatum, Vis: Visual. Inset: enlargement of the somatosensory cortex. (B) Picture of somatosensory cortex and deeper structures from mouse observed through a X4 objective. Notice the two stimulation electrodes positioned 300 μm apart in L4 and the patch clamp recording electrode in L2/3.

Figure 2

Identifying and recording L2/3 pyramidal neuron from the somatosensory cortex (A) Schema of a sagittal section from the mouse brain illustrating the somatosensory cortex (Ss Cx). Hp: Hippocampus, St: Striatum. Notice the two stimulation electrodes on L4 and the patch clamp recording electrode in L2/3. The blue dot line indicates the perpendicular bisector of the segment connecting the stimulations electrodes. (B) Picture of the Ss Cx taken with a 40× immersion objective. Notice the patch clamp electrode positioned on the soma of a putative pyramidal cell. (C) Fluorescent picture showing the same cell as in B recorded in whole-cell configuration. Notice the single apical dendrite projecting towards the cortical surface and multiple basal dendrites pointing laterally.

Figure 3

Confirmation of independence of the two sets of axons stimulated in L4 (A) Schema of the experimental protocol. Stimulation of two independent sets of L4 excitatory axons that activate feedforward inhibition microcircuits in L2/3. (B) Voltage clamp recording of a L2/3 pyramidal neuron in response to the stimulation of the test and conditioning pathways at the minimal intensity necessary for inducing five consecutive EPSCs (1 Th.). 10 s between each stimulation. Vh = −70 mV. (C) EPSCs induced by the test stimulation at 1.2 Th (average of 5 sweeps). (D) EPSCs induced by a train of 10 shocks at 10 Hz at 1.2 Th. Applied on the conditioning pathways followed by a single shock at 1.2 Th. on the test pathway. Notice the depression of EPSCs during repetitive stimulation (blue dot lines). Inset: Superimposition of the test pathways EPSCs evoked before and after the conditioning train (averages of 5 sweeps). Similar time courses and amplitudes indicate the independency of the pathways.

Figure 4

Hyperexcitability of pyramidal neurons in Stxbp1^hap animals compared to Stxbp^wt (A) Schema of the experimental protocol. Recording of the membrane potential of a basket cell in response to L4 stimulation. (B) Response of a basket cell from a Stxbp1^wt animal to the repetitive stimulation of L4 excitatory axons (10 shocks at 10 Hz, 5 superimposed sweeps). The cell keeps firing action potentials during the whole protocol duration. (C) Response of a basket cell from a Stxbp1^hap animal to same stimulation protocol. The cell stops firing APs after few shocks due to the failure of excitatory synapses (dos Santos et al., 2023). (D) Schema of the experimental protocol. Recording of the membrane potential of a L2/3 pyramidal neuron in response to L4 stimulation. (E) Inhibitory synaptic conductance (Gi) of a L2/3 pyramidal neuron from a Stxbp1^wt animal during repetitive stimulation of L4 axons (10 shocks at 10 Hz). Gi was calculated using a method described elsewhere (House et al., 2011; Dos Santos et al., 2023). (F) Same as in (E), from a Stxbp1^hap animal. Inset: superimposition of Gi induced by the last of the 10 shocks for Stxbp1^wt and Stxbp1^hap animals. Notice that 50 ms after the shock (dashed line) Gi is still significant for Stxbp1^wt but not for Stxbp1^hap animals. (G) Schema of the experimental protocol. Recording of a L2/3 pyramidal neuron in response to the stimulation of two independent sets of L4 excitatory axons that activate feedforward inhibition microcircuits in L2/3. (H) Up left: Response of a Stxbp1^wt pyramidal cell to a single stimulation of the test pathway at 90% of Th AP (3 action potentials evoked out of 5 trials). Response of the same neuron to the repetitive stimulation of the conditioning pathway (1.2 Th EPSC) followed 50 ms later by a single shock applied on the test pathway (90% Th AP). Only 1 action potential was evoked out of 5 trials, demonstrating a decrease in excitability. (I) Same set up from a Stxbp1^hap animal. The pyramidal neuron responded by 2 action potentials in control condition and by 3, after the conditioning train. The excitability was not decreased. (J) Paired diagram illustrating the firing probability of the same pyramidal neurons as in I in response to different intensity stimulations applied before and after the conditioning train.