A phenomenon characterized by the experience of seeing light without any light actually entering the retina is called a phosphene. This phenomenon is also known as photopsias and the two terms have been used interchangeably in the literature. The most familiar type of phosphene is the pressure phosphene, which is elicited by pressing on the orbit, e.g. rubbing of the eyes. According to a pupil of Aristotle, it was Alcmaeon of Croton (circa 530–470 BC) who first reported that “the eye obviously has fire within, for when it is stuck this fire flashes out,” [1]. Phosphenes can occur not only through external stimuli, but also through drugs [2], [3], ionizing radiation [4]–[7], electrical [8]–[13], or magnetic stimulation [14]–[19] of the visual cortex, such as via Transcranial Magnetic Stimulation (TMS) [14]–[19]. Meister et al. [20] performed single-pulse TMS on 22 healthy subjects and observed that, with the same intensity TMS, some subjects experienced phosphenes and some didn't. The authors followed up their experiments with fMRI using checkerboard stimuli to investigate neurological differences between phosphene perceivers and non-perceivers. Subjects who did not report phosphenes had slightly greater activation in the primary visual cortex (V1). Subjects who did report phosphenes had greater extra striatal activation such as in V2/V3 areas. The authors concluded that the level of extra striatal activation seemed to be related to phosphene perception whereas the level of striate activation did not play a critical role, suggesting that phosphene perception may be related to the processing of information in V2 and V3 areas rather than V1. A more recent study [21] used simultaneous TMS and fMRI on 12 healthy subjects to record cortical activation induced by TMS. [21]. While all subjects demonstrated widespread cortical activations in response to TMS, subjects who reported seeing phosphenes had more significant and greater areas of activation within their visual cortex including the primary visual cortex [21]. A recent study using simultaneous TMS and electroencephalography (EEG) also concluded that phosphene perception arises after extensive processing in the brain. [22].

In addition to using magnetic stimulation, which induces electrical currents in the brain via changing magnetic fields, direct electrical stimulation of the occipital lobe also elicits phosphenes. Non-invasive electrical stimulation of the occipital lobe, such as transcranial Direct Current Stimulation (tDCS) of the V1 area has also been shown to elicit phosphenes [11], [12]. Furthermore, the non-invasive electrical stimulation technique, transcranial alternating current stimulation (tACS), over the occipital lobe has been shown to induce phosphenes as well [23]. In fact, using tACS has brought up the issue of whether the phosphenes are solely originating from the occipital area or whether there may be a component with retinal origin as well [13]. Schutter and colleagues [13] performed frontal and occipital tACS on 8 healthy volunteers and reported that tACS of the frontal lobe not only evoked phosphenes but that the phosphenes evoked by the frontal stimulation, reported by the same subjects, were significantly more intense than those evoked by occipital stimulation. Schutter et al [13] hypothesized that in the case of frontal lobe stimulation much of the current may have leaked through the scalp into the retina, and the reported phosphenes may have been caused by stimulation of the retina.

Beyond externally induced phosphenes, phosphenes may also occur spontaneously in individuals with diseased retinas, in normal subjects exposed to high-energy radiation [4]–[7] or taking certain medications [2], [3], or during intraocular insults, such as posterior vitreous detachment [24], [25] or in individuals with a range of retinal or CNS diseases, including retinal detachment or inflammatory, ischemic, autoimmune or degenerative disease [26], [27]. An example of fMRI from spontaneously generated phosphenes also presented in a case study of an individual with brain infarct [28]. In retinal degenerative diseases, phosphenes may be the manifestations of spontaneous activity in degenerating retinal cells due to remodeling of the inner retina or to an unregulated overproduction of free radicals that produce bio/chemiluminescent photons [29]. Chemiluminescent photons are then absorbed by the photoreceptors and initiate a photo-transduction cascade, which results in the perception of phosphenes [30]. Narici et al. [31] later revealed that the lipid peroxidation of the photoreceptors can produce (bio)chemiluminescent photons that generate anomalous visual effects, such as those associated with retinal phosphenes [31]. One of the most severe group of retinal degenerative diseases is Leber's Congenital Amaurosis (LCA), a disease characterized by profound impairment of the retina and poor visual function in infancy and early childhood followed by progressive degeneration and loss of retinal cells [32]–[34]. In our studies developing fMRI as an outcome measure for gene therapy intervention in LCA2, we were struck by the fact that many of the patients reported experiencing phosphenes, and a few on a daily basis, which was not revealed in their clinical history. Furthermore, there are no studies investigating fMRI results of patients with retinal degenerative disease reporting phosphenes. In this report we describe, for the first time, fMRI results from several LCA2 patients during episodes of phosphenes and in the absence of any phosphenes. We show that patients who spontaneously see phosphenes have immense and significant activation in their occipital lobes covering all retinotopic visual centers in the brain whereas they show minimal activation (depending on their age and stage of LCA) when they do not experience phosphenes. The results of this study demonstrate the impact that phosphenes have on vision and on fMRI testing in patients with LCA2 and suggest that queries into phosphene experience could be helpful for understanding test-to-test variations in vision testing.

A phenomenon characterized by the experience of seeing light without any light actually entering the eye is called phosphenes or photopsias. Phosphenes can occur spontaneously or via induction by external stimuli. Previous reports regarding phosphenes have primarily focused on externally induced phosphenes from the occipital cortex using magnetic stimulation[14]–[19], alternating [23] or direct current [11], [12]. Beyond externally induced phosphenes, phosphenes may also occur spontaneously [4]–[7], [23]–[26]. Only a hand full of these reports used fMRI to study activations induced by these phenomenon. A more recent study [21] used fMRI on 12 healthy subjects to test the direct relationship between the threshold of TMS and phosphene perceptions. Authors showed a threshold dependence for TMS induced phosphenes and reported significantly greater distribution of cortical activations for subjects who experienced the induced phosphenes as opposed to those who did not [21]. In addition to stimulated phosphenes, fMRI from spontaneous phosphene was presented from a case report on an individual with brain infarct [28].

In a separate self-report study Schutter and Hortensius [13] examined a group of normal controls undergoing frontal and occipital stimulations to compare the intensity of phosphenes created by the retina as compared to those originated from the occipital cortex. Based on the self-report of study participants, phosphenes evoked by the frontal stimulations, which are attributed to retinal origin, were significantly more intense than those evoked by occipital stimulations [13].

In retinal degenerative diseases, phosphenes may be the manifestations of spontaneous activity in degenerating retinal cells. In the current report we present fMRI results from 3/10 individuals with LCA2 who participated in an ongoing gene therapy clinical trial and neuroimaging study and reported experiencing phosphenes on a daily basis. The phosphene episodes for the rest of LCA2 participants (7/10) occurred infrequently and were not reported during fMRI. Functional results are presented when the three individuals were/were not experiencing phosphenes to demonstrate the differences in human brain activity in the presence or absence of these internally generated light experiences.

As illustrated in Figures 3–5, all LCA2 subjects' responses to the checkerboard stimuli, showed an overwhelming amount of cortical activations covering the entire bilateral visual cortices in the presence of phosphenes (panels B and D). In contrast, there were limited cortical activations recorded in the absence of phosphenes (panels A and C). Comparison of the fMRI results for treated (panels C) to the untreated eyes (panels A) in the absence of phosphenes shows noticeably higher amount of significant activations for patients' treated eyes, similar to what we previously reported in other subjects (e.g. CH09, CH13, NP01, CH12) who do not regularly experience phosphenes [34], [37]. The one exception is NP02, who had received a low dose of the gene therapy vector (see Table 1) and whose treated eye activation may have been diminished by the macular hole, though visual function improvements after gene therapy were reported for this subject [32], [35]. fMRI results of all three of the unilaterally injected subjects when experiencing phosphenes clearly show that the magnitude and distribution of cortical activations were by far more extensive, intense and widespread throughout the visual cortices during phosphenes. Such episodes persisted regardless of the extent of retinal disease (including the macular hole in NP02) [32], [35], (Figure 3: Panels B and D). It is important to note that clinical fMRI results reported on CH08 [34] in a separate study involving unilateral administration, was carefully monitored to be free of phosphene episodes.

The cortical activation patterns produced while subjects were experiencing phosphenes are by far much larger and more extensively distributed in the brain than the activation pattern triggered by the same stimuli in normal sighted individuals. This is illustrated in Figure 6 where fMRI results from the left and right eyes of two normally sighted controls are presented. As expected, both normal subjects showed significant and symmetrically distributed bilateral activation. However, the amount and distribution of activation in the normal control subjects are noticeably less than what was observed in patients experiencing phosphenes. Also, as compared to sighted controls, the pattern of cortical activity for the LCA patients in the absence of phosphenes is drastically lower and limited more to the extra striatal areas.

As depicted in Table 1, phosphene experiences did not appear to be dependent on patients' age, gender, visual acuity, RPE65 mutation type, concentration, or volume of vector they received for gene therapy. Although the frequency and characteristics of phosphene experiences were not queried before and after gene therapy, some LCA2 patients reported experiencing them “somewhat” less frequently.

In agreement with widespread cortical activation, all three patients reported interference with their vision while experiencing phosphenes. In fact, all patients stated that they have to wait for phosphene episodes to end before they can continue their activities. Earlier studies in patients with retinitis pigmentosa by Bittner et al [38], [39] also described that patients with frequent occurrence of photopsias or spontaneous phosphenes reported interference with their vision. The results of fMRI presented here (immense cortical activations) may explain the inabilities of subjects to see while experiencing phosphenes (saturated/paralyzed visual cortex). The pattern of cortical activations invoked by phosphenes along with patients' self reports on inability to see during phosphene episodes indicates that patients should be queried about their phosphene experience during clinical testing, as phosphenes could potentially interfere with results of standard measures of visual function (visual acuity, visual field testing). Such effects may explain the challenges of high test-to-test reproducibility in tests of Goldman visual fields in LCA2 patients described by Roman et al. [40]. Thus, similar to the recommendation previously set forth by Bittner et al. [38], [39], our fMRI results also strongly advocate the need for awareness of phosphene experiences in all patients with retinal disease.

Although the cause of phosphenes in LCA2 patients is unknown, we hypothesize that they are of retinal origin and due to their RPE65-related disease. One plausible explanation suggested by Bókkon [41] is that retinal phosphenes may be due to overproduction of the ultra weak spontaneous photons [42] that are constantly emitted from the free radical reactions of retinal cells without any external excitation. If these biophoton emissions exceed a certain threshold, it can appear as phosphenes to the subject.

In summary, phosphenes may occur in a significant proportion of LCA2 patients and the characteristics can take on several different forms and can have an impact on the patient's visual experience. Although we did not perform correlations between visual function and phosphenes, we hope data presented report show not only that phosphenes can impact fMRI results but they also can potentially affect other forms of visual function testing. Future gene therapy clinical trials for retinal degeneration should gather additional information on phosphene experiences and correlate this information with visual function test results. fMRI data also illustrate the potential negative impact that phosphene perception may have on vision testing. fMRI has proven to be a sensitive, non-invasive and reproducible test paradigm for these purposes and can complement standard visual function testing. In this study, fMRI confirmed the long-term effects of retinal gene augmentation therapy in two LCA2 patients who had received medium (CH08 & CH11) vector genome concentrations [35]. Ultimately, the incorporation of fMRI studies with standard retinal function test paradigms will reveal whether gene therapy or other interventions slow or halt the progression of disease and/or improve vision. Results from the present report, however, bring awareness to clinicians and researchers on the effect this phenomenon may have on patients' clinical assessment. Although it should be noted that, as reported by Bittner et al [38], phosphenes may be only one source of increased variability in vision testing and other factors such as patient or operator related variables could also lead to inconsistent test results.

Awareness of potential phosphene phenomena in patients with retinal diseases is particularly important with respect to evaluation of emerging therapies including gene, molecular, and cellular therapies.