A rat model of a focal mosaic expression of PCDH19 replicates human brain developmental abnormalities and behaviours.

Protocadherin 19 gene-related epilepsy or protocadherin 19 clustering epilepsy is an infantile-onset epilepsy syndrome characterized by psychiatric (including autism-related), sensory, and cognitive impairment of varying degrees. Protocadherin 19 clustering epilepsy is caused by X-linked protocadherin 19 protein loss of function. Due to random X-chromosome inactivation, protocadherin 19 clustering epilepsy-affected females present a mosaic population of healthy and protocadherin 19-mutant cells. Unfortunately, to date, no current mouse model can fully recapitulate both the brain histological and behavioural deficits present in people with protocadherin 19 clustering epilepsy. Thus, the search for a proper understanding of the disease and possible future treatment is hampered. By inducing a focal mosaicism of protocadherin 19 expression using in utero electroporation in rats, we found here that protocadherin 19 signalling in specific brain areas is implicated in neuronal migration, heat-induced epileptic seizures, core/comorbid behaviours related to autism and cognitive function.

Genetic interaction between PLK1 and downstream MCPH proteins in the control of centrosome asymmetry and cell fate during neural progenitor division.

Alteration of centrosome function and dynamics results in major defects during chromosome segregation and is associated with primary autosomal microcephaly (MCPH). Despite the knowledge accumulated in the last few years, why some centrosomal defects specifically affect neural progenitors is not clear. We describe here that the centrosomal kinase PLK1 controls centrosome asymmetry and cell fate in neural progenitors during development. Gain- or loss-of-function mutations in Plk1, as well as deficiencies in the MCPH genes Cdk5rap2 (MCPH3) and Cep135 (MCPH8), lead to abnormal asymmetry in the centrosomes carrying the mother and daughter centriole in neural progenitors. However, whereas loss of MCPH proteins leads to increased centrosome asymmetry and microcephaly, deficient PLK1 activity results in reduced asymmetry and increased expansion of neural progenitors and cortical growth during mid-gestation. The combination of PLK1 and MCPH mutations results in increased microcephaly accompanied by more aggressive centrosomal and mitotic abnormalities. In addition to highlighting the delicate balance in the level and activity of centrosomal regulators, these data suggest that human PLK1, which maps to 16p12.1, may contribute to the neurodevelopmental defects associated with 16p11.2-p12.2 microdeletions and microduplications in children with developmental delay and dysmorphic features.

Deficient adaptation to centrosome duplication defects in neural progenitors causes microcephaly and subcortical heterotopias.

Congenital microcephaly (MCPH) is a neurodevelopmental disease associated with mutations in genes encoding proteins involved in centrosomal and chromosomal dynamics during mitosis. Detailed MCPH pathogenesis at the cellular level is still elusive, given the diversity of MCPH genes and lack of comparative in vivo studies. By generating a series of CRISPR/Cas9-mediated genetic KOs, we report here that - whereas defects in spindle pole proteins (ASPM, MCPH5) result in mild MCPH during development - lack of centrosome (CDK5RAP2, MCPH3) or centriole (CEP135, MCPH8) regulators induces delayed chromosome segregation and chromosomal instability in neural progenitors (NPs). Our mouse model of MCPH8 suggests that loss of CEP135 results in centriole duplication defects, TP53 activation, and cell death of NPs. Trp53 ablation in a Cep135-deficient background prevents cell death but not MCPH, and it leads to subcortical heterotopias, a malformation seen in MCPH8 patients. These results suggest that MCPH in some MCPH patients can arise from the lack of adaptation to centriole defects in NPs and may lead to architectural defects if chromosomally unstable cells are not eliminated during brain development.

Single-cell transcriptomics of the early developing mouse cerebral cortex disentangle the spatial and temporal components of neuronal fate acquisition.

In the developing cerebral cortex, how progenitors that seemingly display limited diversity end up producing a vast array of neurons remains a puzzling question. The prevailing model suggests that temporal maturation of progenitors is a key driver in the diversification of the neuronal output. However, temporal constraints are unlikely to account for all diversity, especially in the ventral and lateral pallium where neuronal types significantly differ from their dorsal neocortical counterparts born at the same time. In this study, we implemented single-cell RNAseq to sample the diversity of progenitors and neurons along the dorso-ventral axis of the early developing pallium. We first identified neuronal types, mapped them on the tissue and determined their origin through genetic tracing. We characterised progenitor diversity and disentangled the gene modules underlying temporal versus spatial regulations of neuronal specification. Finally, we reconstructed the developmental trajectories followed by ventral and dorsal pallial neurons to identify lineage-specific gene waves. Our data suggest a model by which discrete neuronal fate acquisition from a continuous gradient of progenitors results from the superimposition of spatial information and temporal maturation.

Modelling genetic mosaicism of neurodevelopmental disorders in vivo by a Cre-amplifying fluorescent reporter.

Genetic mosaicism, a condition in which an organ includes cells with different genotypes, is frequently present in monogenic diseases of the central nervous system caused by the random inactivation of the X-chromosome, in the case of X-linked pathologies, or by somatic mutations affecting a subset of neurons. The comprehension of the mechanisms of these diseases and of the cell-autonomous effects of specific mutations requires the generation of sparse mosaic models, in which the genotype of each neuron is univocally identified by the expression of a fluorescent protein in vivo. Here, we show a dual-color reporter system that, when expressed in a floxed mouse line for a target gene, leads to the creation of mosaics with tunable degree. We demonstrate the generation of a knockout mosaic of the autism/epilepsy related gene PTEN in which the genotype of each neuron is reliably identified, and the neuronal phenotype is accurately characterized by two-photon microscopy.

Influence of heart rate on FFR measurements: An experimental and clinical validation study.

BACKGROUND: Functional lesion assessment in stable coronary disease is considered the gold standard. The result of fractional flow reserve (FFR) in stable coronary disease is often a decision-maker for patient qualification. Taking into account the paramount position of FFR, it is crucial to acknowledge and reduce all potential bias. AIMS: In the present study, we quantified the influence of elevated HR on FFR results using a preclinical model and then validated the results in a clinical setting. METHODS AND RESULTS: The relationship between FFR and HR was first explored experimentally in a porcine model. A clinical validation study was conducted in patients with isolated moderate lesions in the left anterior descending artery (LAD) or right coronary artery (RCA). In both the experimental and clinical arms, FFR was measured at resting HR and with pacing at 100, 130, 160, and 180 (for pigs) beats per minute. In the porcine model and in the clinical settings, a significant correlation between FFR and HR was confirmed in the LAD (r = 0.89, p < .0001; r = 0.53, p = .00002), but not in the RCA (r = -0.19, p = .5; r = 0.14, p = .3). Post hoc analyses revealed that the FFR values in the LAD at 130/min and above tended to be significantly different from the baseline HR. CONCLUSIONS: The results of this study indicate that in an experimental setting, tachycardia might be responsible for an overestimation of FFR results in LAD lesions.

A Localized Scaffold for cGMP Increase Is Required for Apical Dendrite Development.

Studies in cultured neurons have established that axon specification instructs neuronal polarization and is necessary for dendrite development. However, dendrite formation in vivo occurs when axon formation is prevented. The mechanisms promoting dendrite development remain elusive. We find that apical dendrite development is directed by a localized cyclic guanosine monophosphate (cGMP)-synthesizing complex. We show that the scaffolding protein Scribble associates with cGMP-synthesizing enzymes soluble-guanylate-cyclase (sGC) and neuronal nitric oxide synthase (nNOS). The Scribble scaffold is preferentially localized to and mediates cGMP increase in dendrites. These events are regulated by kinesin KifC2. Knockdown of Scribble, sGC-ß1, or KifC2 or disrupting their associations prevents cGMP increase in dendrites and causes severe defects in apical dendrite development. Local cGMP elevation or sGC expression rescues the effects of Scribble knockdown on dendrite development, indicating that Scribble is an upstream regulator of cGMP. During neuronal polarization, dendrite development is directed by the Scribble scaffold that might link extracellular cues to localized cGMP increase.

Evolutionary Gain of Dbx1 Expression Drives Subplate Identity in the Cerebral Cortex.

Changes in transcriptional regulation through cis-regulatory elements are thought to drive brain evolution. However, how this impacts the identity of primate cortical neurons is still unresolved. Here, we show that primate-specific cis-regulatory sequences upstream of the Dbx1 gene promote human-like expression in the mouse embryonic cerebral cortex, and this imparts cell identity. Indeed, while Dbx1 is expressed in highly restricted cortical progenitors in the mouse ventral pallium, it is maintained in neurons in primates. Phenocopy of the primate-like Dbx1 expression in mouse cortical progenitors induces ectopic Cajal-Retzius and subplate (SP) neurons, which are transient populations playing crucial roles in cortical development. A conditional expression solely in neurons uncouples mitotic and postmitotic activities of Dbx1 and exclusively promotes a SP-like fate. Our results highlight how transcriptional changes of a single fate determinant in postmitotic cells may contribute to the expansion of neuronal diversity during cortical evolution.

Synaptogenesis Stimulates a Proteasome-Mediated Ribosome Reduction in Axons.

Ribosomes and a subset of cellular mRNAs are trafficked into axons of developing neurons. The axonal localization of translational machinery allows new proteins to be rapidly and locally synthesized during axonal growth and pathfinding. However, in mature neurons, axonal ribosomes are significantly reduced or even absent. The mechanism that elicits this removal is currently unknown. Here, we demonstrate that synapse formation is the trigger for ribosome reduction in mature axons. In vivo analysis shows that axonal ribosome levels decrease in rat brain at a developmental stage coincident with synapse formation. Next, we observe in vitro that different synaptogenic inducers trigger an overall decrease of ribosomal proteins and rRNA in the axons of spinal motor neurons. We further observe that this process is dependent on the ubiquitin-proteasome system but not on autophagy. Together, these data identify synaptogenesis as the long missing biological trigger that leads to ribosome disappearance during axonal maturation.

The development of synaptic transmission is time-locked to early social behaviors in rats.

The development of functional synapses is a sequential process preserved across many brain areas. Here, we show that glutamatergic postsynaptic currents anticipated GABAergic currents in Layer II/III of the rat neocortex, in contrast to the pattern described for other brain areas. The frequencies of both glutamatergic and GABAergic currents increased abruptly at the beginning of the second postnatal week, supported by a serotonin upsurge. Integrative behaviors arose on postnatal day (P)9, while most motor and sensory behaviors, which are fundamental for pup survival, were already in place at approximately P7. A reduction in serotonin reuptake accelerated the development of functional synapses and integrative huddling behavior, while sparing motor and sensory function development. A decrease in synaptic transmission in Layer II/III induced by a chemogenetic approach only inhibited huddling. Thus, precise developmental sequences mediate early, socially directed behaviors for which neurotransmission and its modulation in supragranular cortical layers play key roles.

In vivo methods for acute modulation of gene expression in the central nervous system.

Accurate and timely expression of specific genes guarantees the healthy development and function of the brain. Indeed, variations in the correct amount or timing of gene expression lead to improper development and/or pathological conditions. Almost forty years after the first successful gene transfection in in vitro cell cultures, it is currently possible to regulate gene expression in an area-specific manner at any step of central nervous system development and in adulthood in experimental animals in vivo, even overcoming the very poor accessibility of the brain. Here, we will review the diverse approaches for acute gene transfer in vivo, highlighting their advantages and disadvantages with respect to the efficiency and specificity of transfection as well as to brain accessibility. In particular, we will present well-established chemical, physical and virus-based approaches suitable for different animal models, pointing out their current and future possible applications in basic and translational research as well as in gene therapy.

Freedom from pulmonary vein stenosis after multiple applications of epicardial ablation energy.

OBJECTIVES: In patients undergoing cardiac surgical procedures, pulmonary vein isolation may be easily accomplished, and it is important to achieve bidirectional conduction block across created lesions. The primary aim of this study was to assess the risk of pulmonary vein stenosis (PVS) after multiple applications of epicardial bipolar radiofrequency energy. METHODS: Thirty-five consecutive patients who were referred for off-pump coronary revascularization with concomitant pulmonary vein isolation and left atrial appendage occlusion were prospectively included in the study. The ablation protocol provided 8 standard epicardial applications of bipolar energy with additional applications until the acute bidirectional conduction block was achieved. Three to 6 months after surgery, patients underwent computed tomography to assess PVS. RESULTS: In all patients, bidirectional conduction block was achieved across the created lesions. In 31 (89%) patients, conduction block was accomplished after the standard 8 energy applications on each side. In 4 (11%) patients, additional applications of energy were needed. All patients had computed tomography (128 total pulmonary veins) scans, which showed no evidence of PVS. CONCLUSIONS: Multiple applications of bipolar radiofrequency energy during off-pump epicardial pulmonary vein isolation did not lead to PVS. Creating bidirectional conduction block using multiple energy applications through created lesions is feasible in all patients using the ablation protocol described.

Intraoperative epicardial focal pulmonary venous electrocardiography in patients with atrial fibrillation.

OBJECTIVES: In patients referred to off-pump coronary artery bypass grafting, pulmonary vein isolation (PVI) may be used for those with persistent atrial fibrillation (AF), an alternative to the Maze procedure. However, the success rate of PVI in persistent AF is limited. The study assesses the prognostic value of focal epicardial electrocardiography of the pulmonary veins (PVs) for surgical ablation results. METHODS: We mapped 140 PV in 35 cases undergoing off-pump coronary artery bypass grafting. Data obtained using a sensing-pacing probe before ablation were analysed. The composite study end-point consisted of the need for electrical cardioversion for in-hospital recurrence of AF and the presence of AF at hospital discharge and after 6 months follow-up confirmed by 24-h Holter electrocardiographic monitoring. RESULTS: In patients with epicardial far-field (FF) signals recorded over at least 1 PV, the composite end-point occurred in 61% (14) vs 25% (3) of patients with no FF signal recorded over any PV (P = 0.04). The presence of FF signals in at least 1 PV significantly increased the risk of composite end-point occurrence (odds ratio 3; P = 0.04). The composite end-point occurred in 86% (6) of patients with FF signals recorded over all PVs and in 39% (11) in the remainder of the study population (P = 0.03). CONCLUSIONS: Intraoperative epicardial focal electrocardiography of PVs revealed more than 40% of PVs had only FF atrial signals. The presence of FF signals in PVs is related to a lower early effectiveness of PVI on ablating AF. Epicardial focal electrocardiography of PVs may be a clinically effective intraoperative tool in the decision-making process between less invasive PVI and the standard Maze procedure.

The female epilepsy protein PCDH19 is a new GABAAR-binding partner that regulates GABAergic transmission as well as migration and morphological maturation of hippocampal neurons.

The PCDH19 gene (Xp22.1) encodes the cell-adhesion protein protocadherin-19 (PCDH19) and is responsible for a neurodevelopmental pathology characterized by female-limited epilepsy, cognitive impairment and autistic features, the pathogenic mechanisms of which remain to be elucidated. Here, we identified a new interaction between PCDH19 and GABAA receptor (GABAAR) alpha subunits in the rat brain. PCDH19 shRNA-mediated downregulation reduces GABAAR surface expression and affects the frequency and kinetics of miniature inhibitory postsynaptic currents (mIPSCs) in cultured hippocampal neurons. In vivo, PCDH19 downregulation impairs migration, orientation and dendritic arborization of CA1 hippocampal neurons and increases rat seizure susceptibility. In sum, these data indicate a role for PCDH19 in GABAergic transmission as well as migration and morphological maturation of neurons.

The X-Linked Intellectual Disability Protein IL1RAPL1 Regulates Dendrite Complexity.

Mutations and deletions of the interleukin-1 receptor accessory protein like 1 (IL1RAPL1) gene, located on the X chromosome, are associated with intellectual disability (ID) and autism spectrum disorder (ASD). IL1RAPL1 protein is located at the postsynaptic compartment of excitatory synapses and plays a role in synapse formation and stabilization. Here, using primary neuronal cultures and Il1rapl1-KO mice, we characterized the role of IL1RAPL1 in regulating dendrite morphology. In Il1rapl1-KO mice we identified an increased number of dendrite branching points in CA1 and CA2 hippocampal neurons associated to hippocampal cognitive impairment. Similarly, induced pluripotent stem cell-derived neurons from a patient carrying a null mutation of the IL1RAPL1 gene had more dendrites. In hippocampal neurons, the overexpression of full-length IL1RAPL1 and mutants lacking part of C-terminal domains leads to simplified neuronal arborization. This effect is abolished when we overexpressed mutants lacking part of N-terminal domains, indicating that the IL1RAPL1 extracellular domain is required for regulating dendrite development. We also demonstrate that PTPδ interaction is not required for this activity, while IL1RAPL1 mediates the activity of IL-1ß on dendrite morphology. Our data reveal a novel specific function for IL1RAPL1 in regulating dendrite morphology that can help clarify how changes in IL1RAPL1-regulated pathways can lead to cognitive disorders in humans.SIGNIFICANCE STATEMENT Abnormalities in the architecture of dendrites have been observed in a variety of neurodevelopmental, neurodegenerative, and neuropsychiatric disorders. Here we show that the X-linked intellectual disability protein interleukin-1 receptor accessory protein like 1 (IL1RAPL1) regulates dendrite morphology of mice hippocampal neurons and induced pluripotent stem cell-derived neurons from a patient carrying a null mutation of IL1RAPL1 gene. We also found that the extracellular domain of IL1RAPL1 is required for this effect, independently of the interaction with PTPδ, but IL1RAPL1 mediates the activity of IL-1ß on dendrite morphology. Our data reveal a novel specific function for IL1RAPL1 in regulating dendrite morphology that can help clarify how changes in IL1RAPL1-regulated pathways can lead to cognitive disorders in humans.

Left Atrial Ganglionated Plexi Detection is Related to Heart Rate and Early Recurrence of Atrial Fibrillation after Surgical Ablation.

INTRODUCTION:: Left atrial ganglionated plexi ablation is an adjuvant technique used to increase the success rate of surgical ablation of atrial fibrillation. Ganglionated plexi ablation requires previous detection. We aimed to assess determinants of successful ganglionated plexi detection and to correlate range of ganglionated plexi ablation with risk of early atrial fibrillation recurrence. METHODS:: The study involved 34 consecutive patients referred for surgical coronary revascularization with concomitant atrial fibrillation ablation. Ganglionated plexi detection was done by inducing vagal reflexes in the area of the pulmonary veins and left atrial fat pads. RESULTS:: Detection of GP was successful in 85% of the patients. There was no difference in preoperative characteristics nor in atrial fibrillation type between patients in whom ganglionated plexi detection was successful and others. The number of detected ganglionated plexi correlated significantly only with preoperative resting heart rate. Significant negative correlation was found in patients with preoperative heart rate>75 beat/min in terms of total number of detected ganglionated plexi (P=0.04). Average number of detected ganglionated plexi was significantly higher in patients with in-hospital atrial fibrillation recurrence requiring electrical cardioversion (3.8±3) in comparison to rest of the study population (2±1.3; P=0.02). In patients in whom 4 or more ganglionated plexi were detected, significantly increased risk of in-hospital atrial fibrillation recurrence was observed (OR 15; 95% CI 1.5-164; P=0.003). CONCLUSION:: Left atrial ganglionated plexi detection was unsuccessful in a considerable percentage of patients. Preoperative heart rate significantly influenced positive ganglionated plexi detection and number of ablated ganglia. Higher number of detected ganglionated plexi was related with early recurrence of atrial fibrillation.

Left atrial ganglionated plexi detection is related to heart rate and early recurrence of atrial fibrillation after surgical ablation

Abstract INTRODUCTION: Left atrial ganglionated plexi ablation is an adjuvant technique used to increase the success rate of surgical ablation of atrial fibrillation. Ganglionated plexi ablation requires previous detection. We aimed to assess determinants of successful ganglionated plexi detection and to correlate range of ganglionated plexi ablation with risk of early atrial fibrillation recurrence. METHODS: The study involved 34 consecutive patients referred for surgical coronary revascularization with concomitant atrial fibrillation ablation. Ganglionated plexi detection was done by inducing vagal reflexes in the area of the pulmonary veins and left atrial fat pads. RESULTS: Detection of GP was successful in 85% of the patients. There was no difference in preoperative characteristics nor in atrial fibrillation type between patients in whom ganglionated plexi detection was successful and others. The number of detected ganglionated plexi correlated significantly only with preoperative resting heart rate. Significant negative correlation was found in patients with preoperative heart rate>75 beat/min in terms of total number of detected ganglionated plexi (P=0.04). Average number of detected ganglionated plexi was significantly higher in patients with in-hospital atrial fibrillation recurrence requiring electrical cardioversion (3.8±3) in comparison to rest of the study population (2±1.3; P=0.02). In patients in whom 4 or more ganglionated plexi were detected, significantly increased risk of in-hospital atrial fibrillation recurrence was observed (OR 15; 95% CI 1.5-164; P=0.003). CONCLUSION: Left atrial ganglionated plexi detection was unsuccessful in a considerable percentage of patients. Preoperative heart rate significantly influenced positive ganglionated plexi detection and number of ablated ganglia. Higher number of detected ganglionated plexi was related with early recurrence of atrial fibrillation.

Right atrium positioning for exposure of right pulmonary veins during off-pump atrial fibrillation ablation.

OBJECTIVES: Concomitant surgical ablation of atrial fibrillation (AF) is recommended for patients undergoing off-pump coronary revascularization in the presence of this arrhythmia. Achievement of optimal visualization of pulmonary veins while maintaining stable haemodynamic conditions is crucial for proper completion of the ablation procedure. This study evaluates the safety and feasibility of right atrial positioning using a suction-based cardiac positioner as opposed to compressive manoeuvres for exposure during off-pump surgical ablation for AF. METHODS: Thirty-four consecutive patients underwent pulmonary vein isolation, ganglionated plexi ablation and left atrial appendage occlusion during off-pump coronary artery bypass grafting. Right atrial suction positioning was used to visualize right pulmonary veins. Safety and feasibility end points were analysed intraoperatively and in the early postoperative course. RESULTS: In all patients, right atrial positioning created optimal conditions to complete transverse and oblique sinus blunt dissection, correct placement of a bipolar ablation probe, detection and ablation of ganglionated plexi and conduction block assessment. In all patients, this entire right-sided ablation procedure was completed with a single exposure manoeuvre. Feasibility end points were achieved in all study patients. CONCLUSIONS: This report documents the safety and feasibility of right atrial exposure using a suction-based cardiac positioner to complete ablation for AF concomitant with off-pump coronary revascularization. This technique may be widely adopted to create stable haemodynamic conditions and optimal visualization of the right pulmonary veins.

Association of Central Sleep Apnea with Impaired Heart Structure and Cardiovascular Hemodynamics in Patients with Chronic Heart Failure.

BACKGROUND Advanced heart failure (HF) is commonly accompanied by central sleep apnea (CSA) with Cheyne-Stokes respiration (CSR). The aim of this study was to evaluate the relationship between CSA/CSR and other clinical features of HF, with particular emphasis on cardiovascular hemodynamics. MATERIAL AND METHODS In 161 stable HF patients with left ventricular ejection fraction (LVEF) ≤45% (NYHA class I-III; mean LVEF 32.8%) the clinical evaluation included: LVEF; left and right ventricular end-diastolic diameter (LVDd, RVDd); ratio of early transmitral flow velocity to early diastolic septal mitral annulus velocity (E/e') assessed by echocardiography; stroke index (SI); heart rate (HR); cardiac index (CI); and systemic vascular resistance index (SVRI) assessed by impedance cardiography (ICG). The comparison was performed between 2 subgroups: one with moderate/severe CSA/CSR - CSR_ [+] (n=51), and one with mild or no CSA/CSR - CSR_ [-] (n=110). RESULTS CSR_ [+] patients presented more advanced NYHA class (p<0.001) and more frequently had permanent atrial fibrillation (p=0.018). Moreover, they had: lower LVEF (p<0.0001); higher LVDd (p<0.0001), RVDd (p<0.001), and E/e' (p<0.001); lower SI (p<0.001) and CI (p=0.009); and higher HR (p=0.044) and SVRI (p=0.016). The following predictors of CSR_ [+] were identified: NYHA class (OR=3.34 per class, p<0.001, which was the only independent predictor); atrial fibrillation (OR=2.29, p=0.019); RV enlargement (OR=2.75, p=0.005); LVEF<35% (OR=3.38, p=0.001); E/e' (OR=3.15; p=0.003); and SI<35 ml/m2 (OR=2.96, p=0.003). CONCLUSIONS Presence of CSA/CSR in HF is associated with NYHA class, atrial fibrillation and more advanced impairment of cardiovascular structure and hemodynamics. Patient functional state remains the main determinant of CSR.

Targeted in vivo genetic manipulation of the mouse or rat brain by in utero electroporation with a triple-electrode probe.

This protocol is an extension to:Nat. Protoc. 1, 1552-1558 (2006); doi:10.1038/nprot.2006.276; published online 9 November 2006This article describes how to reliably electroporate with DNA plasmids rodent neuronal progenitors of the hippocampus; the motor, prefrontal and visual cortices; and the cerebellum in utero. As a Protocol Extension article, this article describes an adaptation of an existing Protocol and offers additional applications. The earlier protocol describes how to electroporate mouse embryos using two standard forceps-type electrodes. In the present protocol, additional electroporation configurations are possible because of the addition of a third electrode alongside the two standard forceps-type electrodes. By adjusting the position and polarity of the three electrodes, the electric field can be directed with great accuracy to different neurogenic areas. Bilateral transfection of brain hemispheres can be achieved after a single electroporation episode. Approximately 75% of electroporated embryos survive to postnatal ages, and depending on the target area, 50-90% express the electroporated vector. The electroporation procedure takes 1 h 35 min. The protocol is suitable for the preparation of animals for various applications, including histochemistry, behavioral studies, electrophysiology and in vivo imaging.