Home video prediction of epileptic vs. nonepileptic seizures in US veterans.

OBJECTIVE: Using video-EEG (v-EEG) diagnosis as a gold standard, we assessed the predictive diagnostic value of home videos of spells with or without additional limited demographic data in US veterans referred for evaluation of epilepsy. Veterans, in particular, stand to benefit from improved diagnostic tools given higher rates of PNES and limited accessibility to care. METHODS: This was a prospective, blinded diagnostic accuracy study in adults conducted at the Houston VA Medical Center from 12/2015-06/2019. Patients with a definitive diagnosis of epileptic seizures (ES), psychogenic nonepileptic seizures (PNES), or physiologic nonepileptic events (PhysNEE) from v-EEG monitoring were asked to submit home videos. Four board-certified epileptologists blinded to the original diagnosis formulated a diagnostic impression based upon the home video review alone and video plus limited demographic data. RESULTS: Fifty patients (30 males; mean age 47.7 years) submitted home videos. Of these, 14 had ES, 33 had PNES, and three had PhysNEE diagnosed by v-EEG. The diagnostic accuracy by video alone was 88.0%, with a sensitivity of 83.9% and specificity of 89.6%. Providing raters with basic patient demographic information in addition to the home videos did not significantly improve diagnostic accuracy when comparing to reviewing the videos alone. Inter-rater agreement between four raters based on video was moderate with both videos alone (kappa = 0.59) and video plus limited demographic data (kappa = 0.60). SIGNIFICANCE: This study demonstrated that home videos of paroxysmal events could be an important tool in reliably diagnosing ES vs. PNES in veterans referred for evaluation of epilepsy when interpreted by experts. A moderate inter-rater reliability was observed in this study.

Clinical identification of psychogenic nonepileptic events using combinations of psychological tests in a veteran sample.

OBJECTIVE: Patients with psychogenic nonepileptic events (PNEE) exhibit heterogenous symptoms and are best diagnosed with long-term video-electroencephalogram (vEEG) data. While extensive univariate data suggest psychological tests may confirm the etiology of PNEE, the multivariate discriminant utility of psychological tests is less clear. The current study aggregated likelihood ratios of multiple psychological tests to evaluate incremental and discriminant utility for PNEE. METHODS: Veterans with vEEG-diagnosed PNEE (n = 166) or epileptic seizures (n = 92) completed self-report measures and brief neuropsychological evaluations during the 4-day vEEG hospitalization. Receiver operating characteristic (ROC) curves identified discriminating psychological tests and corresponding cut-scores (0.85 minimum specificity). Likelihood ratios from the remaining cut-scores were sequentially linked using the sample base rate of PNEE (64%) and alternative base rates (10%, 20%, 30%, 40%) to estimate posttest probabilities (PTP) of test combinations. RESULTS: The Health Attitudes Survey, Health History Checklist, and Minnesota Multiphasic Personality Inventory-2-Restructured Form scales FBS-r, RC1, MLS, and NUC were identified as discriminating indicators of PNEE. Average PTPs were ≥90% when three or more indicators out of six administered were present at the sample base rate. Regardless of PNEE base rate, PTP for PNEE was ≥98% when all discriminating indicators were present and 92-99% when five of six indicators administered were present. PTPs were largely consistent with observed positive predictive values, particularly as indicators present increased. SIGNIFICANCE: Aggregating psychological tests identified PNEE with a high degree of accuracy, regardless of PNEE base rate. Combining psychological tests may be useful for confirming the etiology of PNEE.

Differentiation of Epileptic and Psychogenic Nonepileptic Seizures Using Single-Channel Surface Electromyography.

PURPOSE: Epileptic seizures (ES) and psychogenic nonepileptic seizures (PNES) are difficult to differentiate when based on a patient's self-reported symptoms. This study proposes review of objective data captured by a surface electromyography (sEMG) wearable device for classification of events as ES or PNES. This may help clinicians accurately identify ES and PNES. METHODS: Seventy-one subjects were prospectively enrolled across epilepsy monitoring units at VA Epilepsy Centers of Excellence. Subjects were concomitantly monitored using video EEG and a wearable sEMG epilepsy monitor, the Sensing Portable sEmg Analysis Characterization (SPEAC) System. Three epileptologists independently classified ES and PNES that contained upper extremity motor activity based on video EEG. The sEMG data from those events were automatically processed to provide a seizure score for event classification. After brief training (60 minutes), the sEMG data were reviewed by a separate group of four epileptologists to independently classify events as ES or PNES. RESULTS: According to video EEG review, 17 subjects experienced 34 events (15 ES and 19 PNES with upper extremity motor activity). The automated process correctly classified 87% of ES (positive predictive value = 88%, negative predictive value = 76%) and 79% of PNES, and the expert reviewers correctly classified 77% of ES (positive predictive value = 94%, negative predictive value = 84%) and 96% of PNES. The automated process and the expert reviewers correctly classified 100% of tonic-clonic seizures as ES, and 71 and 50%, respectively, of non-tonic-clonic ES. CONCLUSIONS: Automated and expert review, particularly in combination, of sEMG captured by a wearable seizure monitor (SPEAC System) may be able to differentiate ES (especially tonic-clonic) and PNES with upper extremity motor activity.

Assessment of the Predictive Value of Outpatient Smartphone Videos for Diagnosis of Epileptic Seizures.

Importance: Misdiagnosis of epilepsy is common. Video electroencephalogram provides a definitive diagnosis but is impractical for many patients referred for evaluation of epilepsy. Objective: To evaluate the accuracy of outpatient smartphone videos in epilepsy. Design, Setting, and Participants: This prospective, masked, diagnostic accuracy study (the OSmartViE study) took place between August 31, 2015, and August 31, 2018, at 8 academic epilepsy centers in the United States and included a convenience sample of 44 nonconsecutive outpatients who volunteered a smartphone video during evaluation and subsequently underwent video electroencephalogram monitoring. Three epileptologists uploaded videos for physicians from the 8 epilepsy centers to review. Main Outcomes and Measures: Measures of performance (accuracy, sensitivity, specificity, positive predictive value, and negative predictive value) for smartphone video-based diagnosis by experts and trainees (the index test) were compared with those for history and physical examination and video electroencephalogram monitoring (the reference standard). Results: Forty-four eligible epilepsy clinic outpatients (31 women [70.5%]; mean [range] age, 45.1 [20-82] years) submitted smartphone videos (530 total physician reviews). Final video electroencephalogram diagnoses included 11 epileptic seizures, 30 psychogenic nonepileptic attacks, and 3 physiologic nonepileptic events. Expert interpretation of a smartphone video was accurate in predicting a video electroencephalogram monitoring diagnosis of epileptic seizures 89.1% (95% CI, 84.2%-92.9%) of the time, with a specificity of 93.3% (95% CI, 88.3%-96.6%). Resident responses were less accurate for all metrics involving epileptic seizures and psychogenic nonepileptic attacks, despite greater confidence. Motor signs during events increased accuracy. One-fourth of the smartphone videos were correctly diagnosed by 100% of the reviewing physicians, composed solely of psychogenic attacks. When histories and physical examination results were combined with smartphone videos, correct diagnoses rose from 78.6% to 95.2%. The odds of receiving a correct diagnosis were 5.45 times greater using smartphone video alongside patient history and physical examination results than with history and physical examination alone (95% CI, 1.01-54.3; P = .02). Conclusions and Relevance: Outpatient smartphone video review by experts has predictive and additive value for diagnosing epileptic seizures. Smartphone videos may reliably aid psychogenic nonepileptic attacks diagnosis for some people.

Provocative induction of psychogenic nonepileptic seizures: Noninferiority of an induction technique without versus with placebo.

We aim to demonstrate, in a sufficiently powered and standardized study, that the success rate of inducing psychogenic nonepileptic seizures (PNES) without placebo (saline infusion) is noninferior to induction with placebo. The clinical data of 170 consecutive patients with suspected PNES who underwent induction with placebo from January 21, 2009 to March 31, 2013 were pair-matched with 170 consecutive patients with suspected PNES who underwent the same induction technique but without addition of placebo from April 1, 2013 to February 7, 2018 at the same center. The success rates of induction were 79.4% (135/170) without placebo and 73.5% (125/170) with placebo. The difference of these two proportions was 5.9%, with two-sided 95% confidence interval ranging from -3.6% to 15.2%, indicating a non-statistically significant difference. The lower bound of the 95% confidence interval (-3.6%) was above the noninferiority margin (δ = -5%), hence inferring noninferiority of induction without versus with placebo. The greater cumulative induction experiences of the clinician performer (influencing the manner/presentation of induction) may supplant the potential advantage from addition of placebo (the means utilized). Among experienced performers, provocative induction without placebo should be the preferred diagnostic approach, given more ethically acceptable transparency and the noninferior success rate when compared to the same induction technique with placebo.

Change in illness perception is associated with short-term seizure burden outcome following video-EEG confirmation of psychogenic nonepileptic seizures.

OBJECTIVES: We aimed to evaluate whether potential changes in the patient's illness perception can significantly influence short-term seizure burden following video-electroencephalography (EEG) confirmation/explanation of psychogenic nonepileptic seizures (PNES). METHODS: Patients with PNES were dichotomized to two groups based on a five-point Symptom Attribution Scale: (a) those who prior to diagnosis perceived their seizures to be solely ("5") or mainly ("4") physical in origin (physical group) and (b) the remainder of patients with PNES (psychological group). The physical group (n=32), psychological group (n=40), and group with epilepsy (n=26) also completed the Brief Illness Perception Questionnaire (BIPQ) prior to diagnosis, and were followed up at 3months as well as at 6months postdiagnosis. RESULTS: At 3months postdiagnosis, the physical group experienced significantly greater improvement in seizure intensity (p=0.002) and seizure frequency (p=0.016) when compared with the psychological group. The physical group was significantly more likely to have modified their symptom attribution toward a greater psychological role to their seizures (p=0.002), and their endorsement on the BIPQ item addressing "consequences" (How much do your seizures affect your life?) was significantly less severe (p'=0.014) when compared with that of the psychological group and the group with epilepsy. At 6months postdiagnosis, the physical group continued to experience significantly greater improvement in seizure intensity (p=0.007) while their seizure frequency no longer reached significant difference (p=0.078) when compared with the psychological group. The physical group continued to be significantly more likely to have modified their symptom attribution toward a greater psychological role to their seizures (p=0.005), and their endorsement on the BIPQ item addressing "consequences" remained significantly less severe (p'=0.037) when compared with the psychological group and the group with epilepsy. CONCLUSIONS: Among patients with PNES, prediagnosis perception of seizures as "solely" or "mainly" physical in cause may be associated with greater likelihood of early postdiagnosis improvement in seizure burden. Within this physical group postdiagnosis, we uncovered preliminary evidence for significantly greater attribution toward psychological roles in seizures as well as reduction in cognitive distortion surrounding the adverse consequences of seizures. These findings portend particular impact of such changes in illness perception for this group.

Performance on the Test of Memory Malingering is predicted by the number of errors on its first 10 items on an inpatient epilepsy monitoring unit.

OBJECTIVE: Performance validity testing is an increasingly vital component of neuropsychological evaluation, though administration of stand-alone performance validity tests (PVTs) can be time-consuming. As the Test of Memory Malingering (TOMM) is among the most commonly used and researched PVTs, much work has focused on creating abbreviated versions while preserving diagnostic accuracy. A recent addition to this effort, errors on the first 10 items of Trial 1 (TOMMe10), was analyzed for its utility in predicting TOMM performance. METHOD: Subjects were 180 veterans seen on a long-term epilepsy monitoring unit. TOMM learning trials, Word Memory Test (WMT), and WAIS-IV Digit Span (for Reliable Digit Span; RDS) were administered as part of a larger battery. Performance invalidity was classified using established cut scores. Diagnostic classification statistics were calculated predicting TOMM, WMT, and RDS performance, including sensitivity, specificity, receiver operating characteristics (ROC), and positive and negative predictive values for multiple TOMMe10 cut scores. RESULTS: A cut score of ≥2 errors on TOMMe10 yielded the highest sensitivity (.88) while maintaining ≥.90 specificity when predicting TOMM (also supported by ROC analysis). This cut score was also optimal when validated against combinations of PVTs (e.g. two of TOMM, WMT, and RDS; WMT and/or RDS). CONCLUSIONS: TOMMe10 shows great promise in predicting future TOMM performance. In settings where time with patients is at a premium, ≥2 errors on TOMMe10 may be used as an early TOMM discontinue criteria, allowing examiners to use their limited time more effectively. The use of TOMMe10 in settings with varying TOMM failure base rates was discussed.

Psychogenic Non-Epileptic Seizures.

PURPOSE OF REVIEW: In this review, we elucidate the evaluation process involved in the diagnosis of psychogenic non-epileptic seizures (PNES). Minimum clinical criteria required to attain this diagnosis via a staged approach are delineated. The psychological underpinnings and management of PNES from the neurologists' perspective are also explored. RECENT FINDINGS: Helpful clues can be deduced from history-taking, seizure semiology, ictal/peri-ictal physical exam, and ictal/inter-ictal EEG data. No single clinical data point is definitively diagnostic of PNES. Instead, the level of certainty for PNES diagnosis is contingent upon concordance of the composite clinical evidence available. Robust neurologist-patient alliance not only facilitates the evaluation process but can influence therapeutic impact. While diagnosis of PNES can be challenging, this diagnosis can be reliably made upon establishing concordance of the historical, physical exam, and video-EEG findings. Evidence-based treatments are available for patients with PNES. Continued efforts remain necessary to enhance timely diagnosis and interdisciplinary management for patients with PNES.

Diagnosis and Treatment of Nonepileptic Seizures.

PURPOSE OF REVIEW: This article details the evaluation process involved in the diagnosis of psychogenic nonepileptic seizures (PNES). The psychological underpinnings, prognostic factors, and recent treatment advances of PNES are also reviewed. RECENT FINDINGS: The diagnosis of PNES is determined based on concordance of the composite evidence available, including historical and physical examination findings, seizure symptoms and signs, and ictal/interictal EEG. No single clinical data point is definitively diagnostic of PNES. The diagnosis of PNES can be challenging at times, such as when seizure documentation on video-EEG cannot be readily obtained. Yet, delayed diagnosis of PNES portends poor outcome. A multicomponent approach to the diagnosis of PNES, with use of an aggregate of available evidence, may facilitate diagnosis and then care of patients with PNES. Emerging evidence supports the effectiveness of cognitive-behavioral-based therapy in the treatment of these patients. SUMMARY: The diagnosis of PNES can be made reliably, and evidence-based treatment now exists. Continued efforts remain necessary to enhance prompt recognition and interdisciplinary management for patients with PNES.

Health resource utilization among US veterans with psychogenic nonepileptic seizures: A comparison before and after video-EEG monitoring.

OBJECTIVE: Prior to establishing the correct diagnosis, patients with psychogenic nonepileptic seizures (PNES) frequently endure significant costs and morbidities associated with utilization of health care resources. In this study of the US veterans population, we aimed to investigate for potential changes in health resource utilization before versus after video-EEG (VEEG) confirmation and disclosure of the PNES diagnosis. METHODS: We prospectively studied 65 veterans with VEEG confirmed diagnosis of PNES, and followed their health care utilization during the subsequent 3 years after the diagnosis. Primary outcomes entailed comparing the quantities of post-VEEG outpatient visits and diagnostic procedures versus those during the 3-year span prior to the diagnosis. Secondary outcome involved specifically the measures of seizure-related antiepileptic drug (AED) use from time points before and after VEEG. RESULTS: Within the category of non-psychiatric outpatient visits, we observed significant post-diagnostic decrease in the utilization of PNES-related outpatient visits (p < 0.001). Contrastingly, we found significant post-diagnostic increase in the utilization of non-PNES-related outpatient visits (p = 0.004). When examining exclusively for psychiatric outpatient visits, we further observed a trend toward increased attendance of outpatient visits (p = 0.056) after VEEG. Utilization of diagnostic procedures was not significantly different before versus after VEEG (p = 0.293). 52.3% of the patients were prescribed AEDs for seizure-related purpose during the one-year period leading up to VEEG. By comparison, only 7.7%, 12.3%, and 10.8% of the patients were still on AEDs for seizure-related purpose at the one-year, two-year, and three-year time points after VEEG, respectively. CONCLUSION: We demonstrate new evidence that VEEG confirmation of the PNES diagnosis among US veterans can significantly reduce key measures of non-psychiatric/PNES-related resource utilization, while also potentially associating with appropriate enhancement of psychiatric outpatient visits. However, our results suggest that within this patient population, further efforts are necessary to address heightened demands for non-PNES-related outpatient visits after VEEG.

Functional neuro-imaging as a pre-surgical tool in epilepsy.

Functional neuro-imaging techniques are helpful in the pre-surgical evaluation of epilepsy for localization of the epileptogenic zone as ancillary tools to electroencephalography (EEG) and magnetic resonance imaging (MRI) or when other localization techniques are normal, non-concordant or discordant. Positron emission tomography (PET) and ictal single photon emission computed tomography (ictal SPECT) imaging are traditional tests that have been reported to have good sensitivity and specificity although the results are better with more expertise as is true for any technique. More recently magnetoencephalogram/magnetic source imaging (MEG/MSI), diffusion tensor imaging and functional magnetic resonance imaging (fMRI) have been used in localization and functional mapping during the pre-surgical work-up of epilepsy. Newer techniques such as fMRI-EEG, functional connectivity magnetic resonance imaging and near infra-red spectroscopy, magnetic resonance spectroscopy and magneto nanoparticles hold promise for further development that could then be applied in the work-up of epilepsy surgery. In this manuscript, we review these techniques and their current position in the pre-surgical evaluation of epilepsy.

Brief group psychoeducation for psychogenic nonepileptic seizures: a neurologist-initiated program in an epilepsy center.

OBJECTIVE: To evaluate therapeutic efficacy upon augmenting the initial communication to patients regarding the diagnosis of psychogenic nonepileptic seizures (PNES) with a novel, brief group psychoeducation administered by the same team that provided the video-electroencephalography (VEEG) confirmed diagnosis and within 4 weeks of the diagnosis. METHODS: Prior to discharge from the epilepsy monitoring unit (EMU), a standardized communication strategy was utilized to explain the diagnosis of PNES to all patients prior to enrollment. Enrolled patients were then randomized to either participation in three successive and monthly group psychoeducational sessions (intervention group), or routine seizure clinic follow-up visits (control group). Both groups completed questionnaires at time of enrollment, and then at approximately 3 months (follow-up 1) and 6 months (follow-up 2) after discharge, assessing for: (1) primary outcomes that include a measure of psychosocial functioning, as well as interval difference in seizure frequency/intensity; and (2) secondary outcomes that include interval seizure-related emergency room visits or hospitalizations, development of new and medically unexplained symptoms, and results of an internal measure of knowledge and perception outcomes. RESULTS: The majority (73%) of patients from the intervention group commenced on therapy sessions within 4 weeks after learning of the diagnosis. Although we did not observe significant group difference in seizure frequency/intensity, patients from the intervention group showed significant improvement on the Work and Social Adjustment Scale (WSAS) scores at both follow-up 1 (p = 0.013) and follow-up 2 (p = 0.038) after discharge from the EMU. In addition, we observed a trend toward lesser likelihood for seizure-related emergency room visits or hospitalizations for the intervention group (p = 0.184), as well as meaningful insights from an internal measure of intervention outcomes. SIGNIFICANCE: These findings suggest that our cost/resource effective, brief group psychoeducational program, when administered early and by the same team who confirmed and communicated the diagnosis of PNES, may contribute to significant functional improvement among participating patients.

Cardiac asystole associated with seizures of right hemispheric onset.

Ictal asystole is frequently underrecognized despite being a potentially lethal condition. We report two cases of ictal asystole with right hemispheric onset. These cases are unique since previous literature reports that seizures associated with bradyarrhythmias typically arise from left hemispheric foci. These cases further underscore the importance of clinical vigilance and the need of an enhanced diagnostic biomarker.

Diagnostic utility of the Structured Inventory of Malingered Symptomatology for identifying psychogenic non-epileptic events.

The Structured Interview of Malingered Symptomatology (SIMS) is a self-report instrument that asks patients whether they experience atypical or implausible symptoms. The instrument has not been evaluated in an epilepsy population, and the potential for it to accurately distinguish between patients with psychogenic non-epileptic events (PNEE) and epileptic event groups has not been established. The SIMS was administered to patients in long-term video-EEG monitoring of these patients, 91 with PNEE and 29 with epilepsy were included in this study. Structured Interview of Malingered Symptomatology total scores as well as neurological and affective subscales were found to be predictors of group membership. Sensitivity and specificity across several different base rates of PNEE as well as maximum level likelihood ratios are presented. The findings not only demonstrate the utility of marked score elevations in differentiating PNEE from epilepsy but also point to considerable caution in interpreting mild elevations. Implications for the utility of this instrument in epilepsy evaluations are discussed.

Intact vs. impaired ictal sensorium: does it affect outcome of psychogenic nonepileptic events following disclosure of diagnosis?

It remains uncertain whether particular ictal manifestations of psychogenic nonepileptic events (PNEE) can reflect distinctive psychological processes or prognostic outcomes. We hypothesize that the integrity of ictal sensorium may affect the clinical outcome of PNEE following disclosure of diagnosis. We prospectively studied 47 veterans who were diagnosed with video-EEG-confirmed PNEE, presented with the diagnosis utilizing a standardized communication strategy, and followed for their clinical progress. When compared to patients with intact ictal sensorium, significantly smaller proportion of patients with impaired ictal sensorium endorsed improvement of either PNEE frequency or intensity across both the initial 1- to 3-month (p=0.005) and ensuing 6- to 9-month (p=0.01) follow-ups. However, improvement among patients with impaired ictal sensorium was more significantly associated with their level of understanding for the PNEE diagnosis across both the initial (rho=0.41, p=0.017) and ensuing (rho=0.43, p=0.015) follow-ups. Our study presents preliminary evidence underscoring the potential clinical significance of ictal sensorial integrity when evaluating patients with PNEE.

The use of all three Test of Memory Malingering trials in establishing the level of effort.

Assessment of the effort level is an essential step in establishing the internal validity of any neuropsychological evaluation. The use of response bias measures as part of a core battery, however, is less common outside of forensic evaluations. The amount of time needed to administer many of these tests is often cited as a likely explanation for their exclusion from routine neuropsychological evaluations. This study examined all three trials of the Test of Memory Malingering (TOMM) in a large sample (n = 213) of inpatients on an epilepsy monitoring unit with the goal of establishing cut scores for early termination. TOMM Trial 1 demonstrated impressive diagnostic accuracy for determining both adequate and suboptimal levels of effort; various cut scores and classification statistics are presented. The optional Retention trial from the TOMM also increased the hit rate 16% in the detection of poor effort. Clinical implications, limitations, and directions for further research are discussed.

Induction of psychogenic nonepileptic events: success rate influenced by prior induction exposure, ictal semiology, and psychological profiles.

PURPOSE: To evaluate whether certain preinduction clinical characteristics may influence the success rate of induction. METHODS: We prospectively enrolled and attempted inductions on 51 patients who were suspected to have psychogenic nonepileptic events based on clinical grounds. In addition to careful examination of the reported ictal semiology, we administered a battery of four psychological instruments to our enrolled patients. KEY FINDINGS: We found that among 42 cases of successful induction, 92.9% (n=39) of these cases were successfully induced on the first attempt (i.e., without prior induction exposure). We observed that induction showed significantly higher rate of success in cases that demonstrate: (1) hypermotor ictal semiology (p=0.029); (2) more prevalent self-reporting of uncommon cognitive and affective symptoms (p=0.035); or (3) higher tendency to rely on coping strategies of "instrumental support" (p=0.013) and "active coping" (p=0.027), when compared to noninducible cases. SIGNIFICANCE: Singular administration of placebo induction on preselected patients with these clinical characteristics may reduce costs by shortening video electroencephalography-(EEG) monitoring sessions and improve the diagnostic yield of video-EEG even for patients with very infrequent events.

Analysis of retinal cell development in chick embryo by immunohistochemistry and in ovo electroporation techniques.

BACKGROUND: Retinal cell development has been extensively investigated; however, the current knowledge of dynamic morphological and molecular changes is not yet complete. RESULTS: This study was aimed at revealing the dynamic morphological and molecular changes in retinal cell development during the embryonic stages using a new method of targeted retinal injection, in ovo electroporation, and immunohistochemistry techniques. A plasmid DNA that expresses the green fluorescent protein (GFP) as a marker was delivered into the sub-retinal space to transfect the chick retinal stem/progenitor cells at embryonic day 3 (E3) or E4 with the aid of pulses of electric current. The transfected retinal tissues were analyzed at various stages during chick development from near the start of neurogenesis at E4 to near the end of neurogenesis at E18. The expression of GFP allowed for clear visualization of cell morphologies and retinal laminar locations for the indication of retinal cell identity. Immunohistochemistry using cell type-specific markers (e.g., Visinin, Xap-1, Lim1+2, Pkcalpha, NeuN, Pax6, Brn3a, Vimentin, etc.) allowed further confirmation of retinal cell types. The composition of retinal cell types was then determined over time by counting the number of GFP-expressing cells observed with morphological characteristics specific to the various retinal cell types. CONCLUSION: The new method of retinal injection and electroporation at E3 - E4 allows the visualization of all retinal cell types, including the late-born neurons, e.g., bipolar cells at a level of single cells, which has been difficult with a conventional method with injection and electroporation at E1.5. Based on data collected from analyses of cell morphology, laminar locations in the retina, immunohistochemistry, and cell counts of GFP-expressing cells, the time-line and dynamic morphological and molecular changes of retinal cell development were determined. These data provide more complete information on retinal cell development, and they can serve as a reference for the investigations in normal retinal development and diseases.