Identifying specific prefrontal neurons that contribute to autism-associated abnormalities in physiology and social behavior

Abstract

Functional imaging and gene expression studies both implicate the medial prefrontal cortex (mPFC), particularly deep-layer projection neurons, as a potential locus for autism pathology. Here, we explored how specific deep-layer prefrontal neurons contribute to abnormal physiology and behavior in mouse models of autism. First, we find that across three etiologically distinct models-in utero valproic acid (VPA) exposure, CNTNAP2 knockout and FMR1 knockout-layer 5 subcortically projecting (SC) neurons consistently exhibit reduced input resistance and action potential firing. To explore how altered SC neuron physiology might impact behavior, we took advantage of the fact that in deep layers of the mPFC, dopamine D2 receptors (D2Rs) are mainly expressed by SC neurons, and used D2-Cre mice to label D2R+ neurons for calcium imaging or optogenetics. We found that social exploration preferentially recruits mPFC D2R+ cells, but that this recruitment is attenuated in VPA-exposed mice. Stimulating mPFC D2R+ neurons disrupts normal social interaction. Conversely, inhibiting these cells enhances social behavior in VPA-exposed mice. Importantly, this effect was not reproduced by nonspecifically inhibiting mPFC neurons in VPA-exposed mice, or by inhibiting D2R+ neurons in wild-type mice. These findings suggest that multiple forms of autism may alter the physiology of specific deep-layer prefrontal neurons that project to subcortical targets. Furthermore, a highly overlapping population-prefrontal D2R+ neurons-plays an important role in both normal and abnormal social behavior, such that targeting these cells can elicit potentially therapeutic effects.

Figure 1.. Prenatal VPA exposure causes deficits in the excitability of mPFC L5 subcortical projection (SC) pyramidal neurons.

A. Retrograde labeling of mPFC L5 SC cells by injection of CTB in ipsilateral mediodorsal (MD) thalamus and labeling of mPFC L5 intratelencephalic projection (IT) neurons by injection of CTB in contralateral mPFC. B. Photomicrographs of injection sites (top, 2x magnification) and labeled cells (bottom, arrowheads). Scale bars = 1 mm and 100 μm. C and D. Current clamp recordings from VPA exposed (purple) and saline control mice (black) in response to current steps in mPFC L5 SC (C) and IT (D) neurons. Examples show responses to −50 and +200 pA steps. Scale bars = 200 ms, 20 mV. VPA = Valproic Acid, SAL = saline control. PL, Prelimbic cortex. IL, Infralimbic cortex.

Figure 2.. Prenatal VPA exposure, FMR1 KO, and CNTNAP2 KO all cause a deficit in the excitability of mPFC L5 subcortical projection (SC) pyramidal neurons, which is associated with decreased input resistance.

A. In mPFC L5 SC neurons, action potential (AP) frequency in response to current injection in autism models (colored circles) compared to controls (black). Background strains are shown in parentheses. B. Same as A, but in IT neurons. C, Input resistance in autism models and controls calculated based on the voltage deflection to a −50 pA current step. Scale bar: 2 mV, 50 pA, 200 ms. D, H current in autism models and controls estimated by the sum of the sag and rebound voltage deflections in response to a −200 pA current step. Scale bar: 2 mV, 250 pA, 500 ms. E, Action potential threshold, halfwidth, and spike frequency adaptation in autism models and controls. ISI: interspike interval. Scale bar: = 500 ms, 50 mV.VPA = Valproic Acid, SAL = saline control, KO = knockout, HET = heterozygote, WT = wild type. PL, Prelimbic cortex. IL, Infralimbic cortex. For C57Bl/6 VPA & SAL, f/i curves (A, B) and input resistance bar graph (C) are re-presented from Fig. 1.

Figure 3.. mPFC D2R+ neurons are persistently activated during social exploration.

Fluorescence in D2R and D1R expressing cells expressing GCaMP6s during social and novel object exploration in wildtype mice. A. Fiber optic implant location for imaging. B. Photomicrograph of fiber tip location (arrowheads) relative to Cre-dependent GCaMP6 expression in mPFC. Scale bar = 100 μm. C. Home cage social exploration (top) and novel object exploration (bottom) assays. D. Left, ΔF/F averaged across mice during social behavior in D1R and D2R-expressing neurons. Right, Quantification of ΔF/F₀ peak and plateau (at +60 seconds averaged over a 20 second window). E. ΔF/F₀ averaged across mice during novel object exploration, with quantifications as in D.

Figure 4.. In VPA mice, mPFC D2R-expressing cells are abnormally activated during social exploration.

mPFC D2R-GCaMP6f fiber photometry during social (top) or novel object (bottom) exploration in C57Bl/6 VPA mice (purple) or saline controls (black). A. Experimental paradigm. B. Average GCaMP6f fluorescence during home cage social (top) or novel object (bottom) exploration assay with the first sniff occurring at t = 0 seconds. C. Peak fluorescence changes (areas bracketed in B) on an expanded time scale. Scale bars = 0.01%, 10 seconds. Dotted line denotes t = 0 s. D. Quantification of peak change in fluorescence following initial sniff and the amplitude of the plateau fluorescence (the mean at t = +60 seconds averaged over a 20 second window).

Figure 5.. Acute optogenetic manipulation of D2R-expressing neurons in the mPFC bidirectionally modulates social exploration behavior in VPA mice.

A. Home cage social and novel object exploration assay coupled with acute optogenetic activation (ChR2) or inactivation (eNpHR) of specific neuronal populations. B. Photomicrograph of fiber tip locations (arrowheads) within bilateral mPFC relative to expression of the fluorescently tagged opsin (in this case, driven by the synapsin promoter). Scale bar = 500 μm. C. In wildtype (left) and VPA (right) mice, acute optogenetic activation (ChR2, yellow) and inactivation (eNpHR, red) of specific mPFC neuron populations during social exploration (top) and novel object exploration (bottom). Each mouse performed the tasks twice, one week apart: once with light ON and once with light OFF. All manipulations were bilateral except for ChR2 stimulation in control mice, which was unilateral. PL, Prelimbic cortex. IL, Infralimbic cortex.