Dgcr8+/- mice are viable, represented at normal birth frequencies in litters, and display gross brain morphologies that are indistinguishable from wild type (WT). We initially sought to confirm that Dgcr8
heterozygosity leads to reduced expression of miRNAs in the cortex, as previously reported in a uniquely generated Dgcr8+/- mouse line [5
]. To assess this, we examined mRNA and miRNA levels in frontal cortex brain lysates from control WT and Dgcr8+/- mice during postnatal development (Figure ). Surprisingly, at postnatal day (P)5 Dgcr8+/- frontal cortices showed no significant changes in Dgcr8
mRNA levels assessed with quantitative PCR (qPCR). qPCR was also used to examine the expression of a panel of select brain-enriched miRNAs and these were similarly unaffected (Figure ). In contrast, by P25, Dgcr8
mRNA was significantly downregulated by 40 ± 9% in Dgcr8+/- cortex (P
= 0.01; Figure ). qPCR established that reduced expression of Dgcr8
mRNA in Dgcr8+/-mice at P25 resulted in the reduced expression of a subset of miRNAs (miR-134, 57 ± 6%, P
= 0.001; miR-491, 61 ± 6%, P
= 0.004; Figure ). These data demonstrate that Dgcr8
heterozygosity leads to reduced biogenesis of miRNAs in cortex; however, this deficiency is not displayed in neonatal mice but rather emerges over development.
Figure 1 Reduced miRNA expression in Dgcr8+/- mice. Dgcr8 mRNA and miRNA expression in the prefrontal cortex of WT (n = 5) and Dgcr8+/- (n = 5) animals at postnatal day (P)5 and P25. (A) Quantitative PCR showing a significant reduction in Dgcr8 mRNA levels in (more ...)
In order to investigate the functional consequences of miRNA deficiency in the brain, we examined the electrophysiological properties of cortical neurons in Dgcr8+/- mice by performing voltage and current clamp recordings on L5 pyramidal neurons in the medial prefrontal cortex (mPFC). These neurons are identifiable by their large soma, prominent apical dendrite (Figure ) and stereotyped electrophysiological properties, including regular spiking activity with minimal accommodation (Figure ). We initially characterized the passive membrane properties and action potential firing capabilities of L5 pyramidal neurons. Input resistance (Rin) was measured through the I-V plot of whole-cell current responses to a series of 5 mV voltage steps (Figure ) and this parameter was significantly increased by approximately 30% in Dgcr8+/- neurons compared to WT (WT Rin = 151 ± 7 MΩ, n = 22 cells; Dgcr8+/- Rin = 195 ± 10 MΩ, n = 20 cells; P = 0.002). Conversely, measurement of the whole-cell capacitance (Cc) showed that this value was significantly decreased in Dgcr8+/- L5 pyramidal neurons (WT Cc = 111 ± 4 pF, n = 24 cells; Dgcr8+/- Cc = 93 ± 4 pF, n = 22 cells; P = 0.004). These changes in passive electrical properties might be attributable to changes in specific membrane conductance or leak currents, so we examined the membrane time constants (τm) and resting membrane potential of L5 pyramidal neurons. τm values were determined by the single exponential fit of the time course of the membrane voltage response to a -25 pA current step, and these values were similar between genotypes (WT τm = 34 ± 2 ms, n = 22 cells; Dgcr8+/- τm = 36 ± 1 ms, n = 27 cells; P = 0.31). The resting membrane potential was also unchanged (WT Vm = -62 ± 2 mV, n = 18 cells; Dgcr8+/- Vm = -61 ± 1 mV, n = 22 cells; P = 0.85). Together, these data show that Dgcr8+/- neurons display altered whole-cell electrical properties, without observable changes to specific membrane properties or leak conductances.
Figure 2 Altered electrical properties, but normal spike firing capabilities of pyramidal neurons in Dgcr8+/- mice. (A) Magnified image (20×) of a neurobiotin-labeled L5 mPFC pyramidal neuron filled during whole-cell recording. (B,C) Representative current-clamp (more ...)
Changes in passive electrical properties might alter neuronal excitability, so we next examined the spike firing capabilities of L5 pyramidal neurons in WT and Dgcr8+/- mice using current-clamp. We first probed neuronal excitability by measuring the minimal current required to elicit an action potential (rheobase current) and this value was not significantly changed (WT = 67 ± 3 pA, n = 22 cells; Dgcr8+/- = 60 ± 3 ms, n = 19 cells; P = 0.08). The threshold for action potential was also not altered (WT VThr = -39 ± 6 mV, n = 23 cells; Dgcr8+/- VThr = -40 ± 1 mV, n = 20 cells). Next we examined action potential firing rates of WT and Dgcr8+/- neurons (Figure ). The input-output relationship was assessed by measuring the neuron's steady-state firing frequency rate as a function of amplitude of injected current (Figure ) and this plot was found to be similar between genotypes. Likewise, examination of the interspike interval of a train of spikes elicited by +200 pA demonstrated indistinguishable firing patterns (Figure ). Together, these data show that despite alterations in passive electrical properties, the excitability and spike firing capabilities of Dgcr8+/- pyramidal neurons is unaffected by reduced miRNA expression.
Because miRNAs can exert powerful regulatory control over translation, we hypothesized that neuronal miRNA deficiency could consequently alter translation-dependent processes that occur during cortical development, including synapse formation and function. To assess this, we used whole-cell patch-clamp electrophysiology to examine synaptic currents in mPFC L5 pyramidal neurons in WT and Dgcr8+/- mice. We analyzed spontaneous excitatory postsynaptic currents (EPSCs; Figure ) and spontaneous inhibitory postsynaptic currents (IPSCs; Figure ) at two time periods during postnatal development. In recordings from P16 to P21 mice (WT n
= 15 cells; Dgcr8+/- n
= 14 cells) we found no significant changes in EPSC event parameters, including amplitude (WT = 13 ± 1 pA; Dgcr8+/- = 13 ± 1 pA; Figure ) or frequency (WT = 2.2 ± 0.3 Hz; Dgcr8+/- = 2.3 ± 0.3 Hz; Figure ). Similarly, IPSC event amplitude (WT = 30 ± 1 pA; Dgcr8+/- = 27 ± 1 pA; Figure ) and frequency (WT = 6.8 ± 0.7 Hz; Dgcr8+/- = 6.5 ± 0.8 Hz; Figure ) were unaffected during the P16 to P21 period. However, when we examined synaptic currents in older P25 to P30 mice (WT n
= 15 cells; Dgcr8+/- n
= 18 cells) we found a significant decrease in the frequency of EPSCs (WT = 3.3 ± 0.5 Hz; Dgcr8+/- = 2.0 ± 0.3 Hz; P
= 0.02; Figure ) without changes in EPSC amplitude (WT = 14.2 ± 0.8 pA; Dgcr8+/- = 13.6 ± 0.8 pA; Figure ), EPSC kinetics (Figure ) or changes in IPSC amplitude (WT = 28 ± 2 pA; Dgcr8+/- = 28 ± 2 pA; Figure ), IPSC frequency (WT = 4.7 ± 0.5 Hz; Dgcr8+/- = 5.1 ± 0.7 Hz; Figure ) or IPSC kinetics (Figure ). To further probe this result, we also examined the 'miniature' EPSC (mEPSC) event population in P25 to P30 mice. Similar to the spontaneous event data, mEPSC frequency was reduced in Dgcr8+/- neurons (WT = 1.9 ± 0.4 Hz; Dgcr8+/- = 0.9 ± 0.1 Hz; P
= 0.02; Additional file 1
) without changes to mEPSC amplitude (WT = 11 ± 1 pA; Dgcr8+/- = 10 ± 1 pA). Lastly, we performed some additional recordings at 33 to 34°C to determine if these deficits persist near physiological temperatures. Elevation of temperatures increased the frequency and amplitude of mEPSC events in both genotypes, and consistent with our previous findings, the deficit in EPSC frequency persisted (WT = 3.8 ± 1.0 Hz; Dgcr8+/- = 2.9 ± 0.9 Hz). Summarily, in WT mPFC we observed endogenous changes in synaptic transmission during maturation, with increased EPSC frequency and decreased IPSC frequency occurring between 3 and 4 postnatal weeks. miRNA deficiency abolished this normal developmental increase in EPSC frequency while not effecting IPSCs, leading to a shift in the balance of excitation/inhibition at P25 to P30. This observed reduction in EPSC frequency in the absence of changes in IPSCs indicates that cortical miRNA deficiency alters the balance of spontaneous synaptic transmission.
Figure 3 Maturation-dependent reduction in excitatory postsynaptic current frequency in Dgcr8+/- mPFC. (A,B) Representative excitatory postsynaptic current (EPSC) recordings from L5 pyramidal neurons from (A) WT and (B) Dgcr8+/- mice. Scale bar = 20 pA, 200 ms. (more ...)
One possible explanation for the neurophysiological changes observed in Dgcr8+/- mice is altered neuron morphology. Input resistance and whole-cell capacitance are functions of membrane area and the primary sites of excitatory synapses onto pyramidal neurons are the dendrites. Accordingly, a decrease in the number of basal dendrites in Dgcr8+/- pyramidal neurons would reduce both membrane area and the number of postsynaptic sites and potentially account for the observed phenotypes. To assess this, we performed Golgi staining (Figure ) and complete three-dimensional reconstructions of L5 pyramidal neurons (Figure ) from WT (n
= 16 cells from 5 animals) and Dgcr8+/- mice (n
= 20 cells from 5 animals). Morphometric analysis (Figure ) of the soma revealed no changes to shape or soma area (WT = 268 ± 13 μm2
; Dgcr8+/- = 245 ± 12 μm2
). Likewise, analysis of the apical dendritic branch showed that these structures remained unaffected in Dgcr8+/- neurons as we found that the number of apical dendrite branch points (WT = 6 ± 1; Dgcr8+/- = 6 ± 1) and the apical dendrite terminal distance from soma (WT = 297 ± 21 μm; Dgcr8+/- = 319 ± 15 μm) were unaltered. However, we found multiple changes to the structure of basal dendrites in Dgcr8+/- mice. Scholl analysis (Figure ) revealed decreased branch complexity in Dgcr8+/- and this was attributable to a decreased number of dendritic branch points (WT = 12 ± 1; Dgcr8+/- = 9 ± 1; P
= 0.02) resulting in a decreased total dendritic length (WT = 1,026 ± 86 μm; Dgcr8+/- = 798 ± 49 μm; P
= 0.02). Lastly, we surveyed dendritic spines and found no differences in spine morphology between genotypes (Additional file 2
) and the spine density on second order branches of the basal dendrites was unchanged (WT = 2.7 ± 0.3 spines/10 μm; Dgcr8+/- = 3.1 ± 0.3 spines/10 μm). Summarily, these data describe a specific morphological deficit in the branching and complexity of basal dendrites of L5 mPFC pyramidal neurons in Dgcr8+/- mice. During postnatal maturation, mPFC basal dendrites undergo elaboration and outgrowth that coincides with the development of the intrinsic electrical properties of pyramidal neurons [16
]. The deficit in basal dendritic branching in Dgcr8+/- mice is consistent with a perturbation to this developmental process and can provide a potential mechanistic explanation for the neurophysiological phenotypes that we describe.
Figure 4 Reduced basal dendritic complexity of L5 pyramidal neurons in Dgcr8+/- mice. Golgi-cox staining of mPFC from WT and Dgcr8+/- mice at P25. (A) Diagram of coronal section of mouse mPFC delineating the area of study and representative 10× and 40× (more ...)