Pyramidal neurons in the medial prefrontal cortical layer 2/3 are an essential contributor to the cellular basis of working memory; thus, changes in their intrinsic excitability critically affect medial prefrontal cortex (mPFC) functional properties. Transient Receptor Potential Melastatin 4 (TRPM4), a calcium-activated nonselective cation channel (CAN), regulates the membrane potential in a calcium-dependent manner. In this study, we uncovered the role of TRPM4 in regulating the intrinsic excitability plasticity of pyramidal neurons in the mouse mPFC layer of 2/3 using a combination of conventional and nystatin perforated whole-cell recordings. Interestingly, we found that TRPM4 is open at resting membrane potential, and its inhibition increases input resistance and hyperpolarizes membrane potential. After high-frequency stimulation, pyramidal neurons increase a calcium-activated non-selective cation current, increase the action potential firing, and the amplitude of the afterdepolarization, these effects depend on intracellular calcium. Furthermore, pharmacological inhibition or genetic silencing of TRPM4 reduces the firing rate and the afterdepolarization after high frequency stimulation. Together, these results show that TRPM4 plays a significant role in the excitability of mPFC layer 2/3 pyramidal neurons by modulating neuronal excitability in a calcium-dependent manner.

Fragile X syndrome (FXS) is frequently associated with auditory hypersensitivity and altered cortical processing, yet the intrinsic ionic mechanisms shaping auditory cortex excitability remain incompletely defined. Here, we tested whether subthreshold conductances contribute to compensatory changes in intrinsic excitability in layer 2/3 (L2/3) pyramidal neurons of the primary auditory cortex (AC) in Fmr1 knockout (Fmr1-KO) mice. We performed whole-cell patch-clamp recordings in acute slices from juvenile mice (P28-P42) and quantified passive properties, firing output, synaptic potentials, and subthreshold currents under pharmacological isolation. Compared with wild type (WT), Fmr1-KO L2/3 neurons displayed a more hyperpolarized resting membrane potential, reduced input resistance, elevated rheobase, prolonged first-spike latency, and reduced firing across depolarizing steps, consistent with a hypoexcitable state. Bath application of BaCl ₂ depolarized the membrane, increased input resistance, and restored firing output and rheobase toward WT levels, indicating that a Ba ²⁺ -sensitive potassium conductance strongly constrains excitability in Fmr1-KO neurons. Voltage-clamp recordings revealed a larger Ba ²⁺ -sensitive inwardly rectifying (Kir-like) current in Fmr1-KO neurons, supporting increased functional Ba ²⁺ -sensitive inwardly rectifying conductance. In contrast, blocking I _h with ZD7288 produced modest changes in passive properties but induced genotype-dependent effects on excitability and enhanced synaptic activity and EPSP summation preferentially in Fmr1-KO neurons, consistent with a role for I _h in input filtering rather than setting basal conductance. Together, these findings identify enhanced Ba ²⁺ -sensitive potassium conductance as a primary determinant of the Fmr1-KO subthreshold conductance state in AC L2/3 pyramidal neurons, suggesting an intrinsic homeostatic mechanism that stabilizes output in the presence of elevated excitatory drive.