In this study, we focused on L2/3 pyramidal neurons, both because (1) in motor cortex, L2/3 is a key node in the local excitatory network, receiving inputs from L3/5A, and providing the major source of excitatory input to L5 (Weiler et al., 2008
), a major cortical output layer (Jones, 1984
); and, (2) we were interested in testing whether MeCP2 deficiency in these neurons induced synaptic abnormalities as previously observed for L5 neurons (Dani et al., 2005
). We used LSPS (Callaway and Katz, 1993
; Katz and Dalva, 1994
) to map excitatory synaptic inputs in MeCP2-deficient neurons. To overcome difficulties in separating primary, or cell-specific, effects of MeCP2 deficiency from downstream signaling abnormalities or homeostatic mechanisms, we used a cell-specific model system of MeCP2 deficiency. An advantage of this cell-specific knockdown approach is the ability to distinguish pre- versus postsynaptic effects of MeCP2 deficiency (in contrast to mutant models in which all neurons lack MeCP2). We generated MeCP2-knockdown neurons (MeCP2kd
) in vivo
, employing RNA interference to down-regulate MeCP2 expression in a sparsely distributed subset of L2/3 pyramidal neurons.
RNAi-mediated knockdown of MeCP2 in motor-frontal L2/3 pyramidal neurons
Previous studies have identified abnormalities in brains of Rett patients and mutant-mouse models of MeCP2-deficiency (Blue et al., 1999
; Dani et al., 2005
; Fukuda et al., 2005
; Asaka et al., 2006
; Chao et al., 2007
; Belichenko et al., 2009
). However, abnormalities revealed by studies of genetic mutations, even when expression is regionally restricted (Chen et al., 2001
; Guy et al., 2001
; Alvarez-Saavedra et al., 2007
) can potentially reflect either cell-specific effects of MeCP2 depletion or compensatory homeostatic mechanisms engaged more globally to counter altered gene expression. To address whether MeCP2 deficiency affects excitatory synaptic input in a cell-specific manner, we used a knockdown strategy in which MeCP2 deficiency was restricted to a small percentage of individual postsynaptic L2/3 pyramidal neurons distributed within a cortical network consisting largely of WT neurons. An aspect of this approach that is also potentially relevant for the pathophysiology of Rett syndrome is that this transfection pattern resembles the mosaic distribution of MeCP2-deficient neurons in the cortex of Rett patients (mostly girls) due to X-chromosome inactivation.
IUEP was performed on embryonic WT mice to transfect individual L2/3 neural progenitor cells with a plasmid encoding GFP and shRNA targeted against MeCP2 (Zhou et al., 2006
) (). Efficient knockdown of MeCP2 expression in GFP-positive neurons was confirmed by immunohistochemistry (), as was a lack of knockdown with a scrambled version of the plasmid (). Although these results do not preclude the possibility of low-level residual MeCP2 expression, together with the mapping studies presented later they do indicate that the knockdown was sufficiently efficient to reduce MeCP2 expression to undetectable levels and to induce circuit abnormalities. GFP expression in transfected neurons remained high at 3–4 weeks, when recordings were made. Cortical thickness was not significantly different compared to unoperated control mice (controls: 1378 ± 21, n = 11 animals; MeCP2kd
: 1336 ± 13 μm, n
= 9 animals). By timing IUEP with respect to the inside-out process of cortical development the transfection was restricted to neurons in L2/3 (). Moreover, only pyramidal neurons were transfected, not inhibitory interneurons (Noctor et al., 2001
; Noctor et al., 2002
; Borrell et al., 2005
The electroporation protocol used here resulted in fairly sparse labeling of L2/3 neurons. We quantified the percentage of labeled L2/3 neurons by counting the number of GFP-positive neurons per high-power field in the transfected region of motor-frontal cortex. On average, 15.5% ± 1.5% of L2/3 neurons expressed GFP (n = 4 animals, 422 cells total). No L5 neurons expressed GFP.
MeCP2kd and WT neurons have similar electrophysiological properties
We recorded from L2/3 neurons in motor-frontal cortex and assessed their intrinsic properties. MeCP2kd and WT neurons did not differ significantly in resting membrane potential (WT: −74 ± 2 mV; MeCP2kd: −72 ± 4 mV), input resistance (WT: 134 ± 10 MΩ; MeCP2kd: 180 ± 26 MΩ), or membrane time constant (WT: 39.2 ± 8.1 msec; MeCP2kd: 33.7 ± 8.8 msec). We further characterized the electrophysiological properties of MeCP2kd and WT pairs of neurons by recording responses in current clamp mode to a family of current steps. Voltage-current relationships for WT and MeCP2kd neurons did not differ significantly (p = 0.50, 2-way ANOVA; ). We also compared the firing properties of WT and MeCP2kd neurons (). Rheobase was indistinguishable (WT: 225 ± 21 pA; MeCP2kd: 217 ± 31 pA), as was the average half-width of action potentials (WT: 1.2 ± 0.1 msec; MeCP2kd: 1.3 ± 0.1 pA). Furthermore, neither f-I relationships (p = 0.47, 2-way ANOVA; ) nor SFA differed between groups (WT: 0.86 ± 0.05; KD: 0.88 ± 0.01). From these tests we concluded that the intrinsic neurophysiological properties of MeCPkd neurons largely resembled those of untransfected neurons.
MeCP2kd neurons have WT-like eletrophysiological properties
Excitatory synaptic input to MeCP2kd neurons is specifically reduced
To map the local sources of excitatory input to these neurons, we used glutamate uncaging and laser scanning photostimulation (LSPS). The basic principles of LSPS circuit mapping are as follows (for reviews, see (Katz and Dalva, 1994
; Shepherd, 2006
)). Caged glutamate is added to the solution bathing a cortical slice, and recording is established on a neuron whose inputs are to be mapped. Photo-release of glutamate by a flash of focused UV light directed to a particular location in the slice activates glutamate receptors on somatodendritic membranes and depolarizes neurons (axons of passage are not activated). Conditions are optimized and calibrated such that individual flashes activate only small numbers of neurons clustered around the center of the beam to fire action potentials. Synaptic inputs from the photostimulated (presynaptic) neurons to the recorded (postsynaptic) neuron are recorded as events in the electrophysiological trace. These events represent the aggregate connectivity from the ~100 or so stimulated neurons onto the recorded neuron. By sequentially sampling hundreds of stimulation sites in a patterned photostimulation grid, a map of the local sources of synaptic inputs converging onto one neuron is generated. The resolution of stimulation (typically ~50–100 μm) is suitable for detailed mapping of inputs on the mesoscopic scale of the local circuit topography (typically 0.5–1.0 mm). For further details, see (Weiler et al., 2008
We used LSPS stimulation parameters previously characterized for this slice preparation (Weiler et al., 2008
). AMPAergic responses were isolated by (1) recording at the GABAergic reversal potential; (2) blocking NMDA receptors; and, (3) rejecting traces contaminated by dendritic responses, arising from stimulation of glutamate receptors on the postsynaptic neuron (Schubert et al., 2001
) (Katz and Dalva, 1994
; Pettit et al., 1999
) (). Dendritic responses only occurred in the vicinity of the somatodendritic region of the neuron and were readily identified by their characteristic short latencies, and were excluded from synaptic input maps (Weiler et al., 2008
LSPS method for mapping excitatory inputs to neighboring transfected and untransfected pairs of neurons
We recorded from L2/3 neurons in motor-frontal cortex, identifying this cortical area by its location anterior to barrel cortex and agranular cytoarchitectonic features (Weiler et al., 2008
; Yu et al., 2008
), readily apparent in these off-sagittal slices (). We targeted nearby (<100 μm) MeCP2kd
and WT neurons, a paradigm enabling direct comparison between neighboring neurons () embedded within the same area of WT cortex, differing in their MeCP2 expression. Neurons were mapped sequentially, as this has been shown to yield results statistically identical to simultaneously mapped pairs (Shepherd and Svoboda, 2005
) and was a more efficient approach in these experiments. Traces that did not contain direct responses () were analyzed to obtain excitatory maps for the neurons in each transfected/untransfected pair.
Excitatory inputs arose from locations directly below the recorded neurons, at approximately the border between L3 and L5A, and from lateral locations in L2/3. For display, these trace arrays were analyzed to generate maps representing the average postsynaptic current over a 50 msec poststimulus time window (). Synaptic input maps recorded in K+- and Cs+-based intracellular solution showed similar amplitude and topographic organization ().
Example maps from neighboring pairs of MeCP2kd and WT pyramidal neurons
To compare excitatory maps of untransfected versus transfected neurons recorded with K+-based intracellular solution (n = 13 pairs), we pooled maps and calculated the average map for each group (). A locus of input was found below the region containing the soma (black pixels), representing ascending L3/5A→2/3 excitatory connections. Computing a vertical laminar profile () showed that the main locus of reduced input occurred 0.4–0.6 mm below the pia, at the L3/5A border. Pixels averaged over this region of interest (ROI) showed a significant reduction in MeCP2kd neurons. These data revealed significantly weaker excitatory inputs for the MeCP2kd neurons (reduced by 33%) to neurons in L2/3 (WT: 11.3 ± 1.7 pA; MeCP2kd: 7.6 ± 1.7 pA) ().
Local sources of synaptic input to transfected/untransfected pairs of L2/3 pyramidal neurons
In contrast to the ascending L3/5A inputs, horizontal inputs to L2/3 neurons, from locations in L2/3 lateral to the recorded neurons, were not significantly different between WT and MeCP2kd (WT: 8.6 ± 2.9 pA; MeCP2kd: 6.6 ± 1.3 pA; ). Therefore, the reduced excitatory synaptic input was apparently specific to the L3/5A→2/3 cortical pathway.
To verify these findings, we performed mapping experiments using a Cs+-based intracellular solution containing 1 mM QX-314 () that provided better voltage control for excitatory maps (presented in this section) and inhibitory maps (presented in the next section). Group analysis of this second set of experiments again showed a locus of input to WT neurons just below the region containing the cell body, confirming the differences observed with K+-based internal solution (). Both the vertical profile and the region average showed significantly reduced input from the L3/5A region (WT: 11.1 ± 1.8 pA; MeCP2kd: 7.1 ± 1.2 pA, 36% reduction) (). As found with K+-based intracellular solution, the L2/3 input was not significantly different between the two groups (WT: 6.2 ± 0.8 pA; MeCP2kd: 8.6 ± 2.2 pA; ), confirming that the synaptic excitatory phenotype induced by MeCP2 deficiency was pathway specific. Because the L2/3 region contained dendritic responses, this region contained fewer data points than the L3/5A region, potentially reducing the power of statistical tests to detect a difference. However, even when the excitatory maps from both K+- and Cs+-based intracellular recordings were pooled, effectively doubling the number of samples for each cell type, the L2/3 input still was not significantly altered in the MeCP2kd neurons (WT: 7.5 ± 1.6 pA; MeCP2kd: 7.4 ± 1.2 pA).
To rule out the potential effects of electroporation, shRNA, and exogenous GFP expression, in separate experiments we transfected animals with a plasmid containing a scrambled version of the shRNA sequence (Zhou et al., 2006
). Excitatory inputs were not significantly different between scrambled versus untransfected neighboring neurons (n
= 11 pairs; ). Although shRNAs can potentially induce an interferon response and synaptic abnormalities including reduced EPSP amplitude (Sledz and Williams, 2004
; Alvarez et al., 2006
), such effects are sequence specific, and the sequence used here was previously shown not to induce dendritic and spine morphological abnormalities when co-transfected with replacement MeCP2 (Zhou et al., 2006
). We therefore interpret the synaptic circuit changes observed here to result specifically from the knockdown of MeCP2.
Scrambled control shows no change in excitatory input to L2/3 neurons
Inhibitory responses are unchanged in MeCP2kd pyramidal neurons
The preceding experiments were all aimed at measuring AMPAergic responses, with the contribution of inhibitory responses minimized by recording at the GABAergic reversal potential. To measure inhibitory inputs, for a subset of the neurons we obtained inhibitory input maps by recording at the reversal potential for glutamatergic responses. In separate experiments, we determined that bath application of the selective GABAA
antagonist SR95531 (gabazine, 10 μM, Tocris) completely blocked these outward currents (n
= 3 WT neurons, ), confirming in this system that these were indeed fast GABAergic events (Brill and Huguenard, 2008
). For recordings with K+
-based intracellular solution, we again excluded perisomatic sites where strong dendritic excitatory conductances accompanied inhibitory inputs. However, for Cs+
-based recordings we included all stimulation sites because dendritic excitatory events were better voltage clamped. Inhibitory maps were similar in both topography and strength between MeCP2kd
neurons and their WT neighbors (K+
= 13 pairs: WT: 46.0 ± 11.9 pA; MeCP2kd
: 47.8 ± 9.5 pA; Cs+
-based: WT: 175.1 ± 36.4 pA; MeCP2kd
: 173.4 ± 35.1 pA) (Fig. 8B–D). These results demonstrate that the reduction in excitatory pathway strength found for the MeCP2kd
neurons was not accompanied by changes in local inhibitory pathways.
Local sources of inhibitory synaptic input