MALDI Imaging Analysis of Neuropeptides in the Africanized Honeybee (Apis mellifera) Brain: Effect of Ontogeny

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Synaptogenesis in the mushroom body calyx during metamorphosis in the honeybee Apis mellifera: An electron microscopic study

The goals of this study are to determine relationships between synaptogenesis and morphogenesis within the mushroom body calyx of the honeybee Apis mellifera and to find out how the microglomerular structure characteristic for the mature calyx is established during metamorphosis. We show that synaptogenesis in the mushroom body calycal neuropile starts in early metamorphosis (stages P1-P3), before the microglomerular structure of the neuropile is established. The initial step of synaptogenesis is characterized by the rare occurrence of distinct synaptic contacts. A massive synaptogenesis starts at stage P5, which coincides with the formation of microglomeruli, structural units of the calyx that are composed of centrally located presynaptic boutons surrounded by spiny postsynaptic endings. Microglomeruli are assembled either via accumulation of fine postsynaptic processes around preexisting presynaptic boutons or via ingrowth of thin neurites of presynaptic neurons into premicroglomeruli, tightly packed groups of spiny endings. During late pupal stages (P8-P9), addition of new synapses and microglomeruli is likely to continue. Most of the synaptic appositions formed there are made by boutons (putative extrinsic mushroom body neurons) into small postsynaptic profiles that do not exhibit presynaptic specializations (putative intrinsic mushroom body neurons). Synapses between presynaptic boutons characteristic of the adult calyx first appear at stage P8 but remain rare toward the end of metamorphosis. Our observations are consistent with the hypothesis that most of the synapses established during metamorphosis provide the structural basis for afferent information flow to calyces, whereas maturation of local synaptic circuitry is likely to occur after adult emergence.

Combining ganglion cell topology and data of patients with glaucoma to determine a structure-function map

PURPOSE: To introduce techniques for deriving a map that relates visual field locations to optic nerve head (ONH) sectors and to use the techniques to derive a map relating Medmont perimetric data to data from the Heidelberg Retinal Tomograph. METHODS: Spearman correlation coefficients were calculated relating each visual field location (Medmont M700) to rim area and volume measures for 10 degrees ONH sectors (HRT III software) for 57 participants: 34 with glaucoma, 18 with suspected glaucoma, and 5 with ocular hypertension. Correlations were constrained to be anatomically plausible with a computational model of the axon growth of retinal ganglion cells (Algorithm GROW). GROW generated a map relating field locations to sectors of the ONH. The sector with the maximum statistically significant (P < 0.05) correlation coefficient within 40 degrees of the angle predicted by GROW for each location was computed. Before correlation, both functional and structural data were normalized by either normative data or the fellow eye in each participant. RESULTS: The model of axon growth produced a 24-2 map that is qualitatively similar to existing maps derived from empiric data. When GROW was used in conjunction with normative data, 31% of field locations exhibited a statistically significant relationship. This significance increased to 67% (z-test, z = 4.84; P < 0.001) when both field and rim area data were normalized with the fellow eye. CONCLUSIONS: A computational model of axon growth and normalizing data by the fellow eye can assist in constructing an anatomically plausible map connecting visual field data and sectoral ONH data.

Intrinsically photosensitive melanopsin retinal ganglion cell contributions to the pupillary light reflex and circadian rhythm

Recently discovered intrinsically photosensitive melanopsin retinal ganglion cells contribute to the maintenance of pupil diameter, recovery and post-illumination components of the pupillary light reflex and provide the primary environmental light input to the suprachiasmatic nucleus for photoentrainment of the circadian rhythm. This review summarises recent progress in understanding intrinsically photosensitive ganglion cell histology and physiological properties in the context of their contribution to the pupillary and circadian functions and introduces a clinical framework for using the pupillary light reflex to evaluate inner retinal (intrinsically photosensitive melanopsin ganglion cell) and outer retinal (rod and cone photoreceptor) function in the detection of retinal eye disease.

The circadian response of intrinsically photosensitive retinal ganglion cells

Intrinsically photosensitive retinal ganglion cells (ipRGC) signal environmental light level to the central circadian clock and contribute to the pupil light reflex. It is unknown if ipRGC activity is subject to extrinsic (central) or intrinsic (retinal) network-mediated circadian modulation during light entrainment and phase shifting. Eleven younger persons (18–30 years) with no ophthalmological, medical or sleep disorders participated. The activity of the inner (ipRGC) and outer retina (cone photoreceptors) was assessed hourly using the pupil light reflex during a 24 h period of constant environmental illumination (10 lux). Exogenous circadian cues of activity, sleep, posture, caffeine, ambient temperature, caloric intake and ambient illumination were controlled. Dim-light melatonin onset (DLMO) was determined from salivary melatonin assay at hourly intervals, and participant melatonin onset values were set to 14 h to adjust clock time to circadian time. Here we demonstrate in humans that the ipRGC controlled post-illumination pupil response has a circadian rhythm independent of external light cues. This circadian variation precedes melatonin onset and the minimum ipRGC driven pupil response occurs post melatonin onset. Outer retinal photoreceptor contributions to the inner retinal ipRGC driven post-illumination pupil response also show circadian variation whereas direct outer retinal cone inputs to the pupil light reflex do not, indicating that intrinsically photosensitive (melanopsin) retinal ganglion cells mediate this circadian variation.

Intrinsically photosensitive melanopsin retinal ganglion cell contributions to the post-illumination pupil response and circadian rhythm

Intrinsically photosensitive retinal ganglion cells (ipRGCs) in the eye transmit the environmental light level, projecting to the suprachiasmatic nucleus (SCN) (Berson, Dunn & Takao, 2002; Hattar, Liao, Takao, Berson & Yau, 2002), the location of the circadian biological clock, and the olivary pretectal nucleus (OPN) of the pretectum, the start of the pupil reflex pathway (Hattar, Liao, Takao, Berson & Yau, 2002; Dacey, Liao, Peterson, Robinson, Smith, Pokorny, Yau & Gamlin, 2005). The SCN synchronizes the circadian rhythm, a cycle of biological processes coordinated to the solar day, and drives the sleep/wake cycle by controlling the release of melatonin from the pineal gland (Claustrat, Brun & Chazot, 2005). Encoded photic input from ipRGCs to the OPN also contributes to the pupil light reflex (PLR), the constriction and recovery of the pupil in response to light. IpRGCs control the post-illumination component of the PLR, the partial pupil constriction maintained for > 30 sec after a stimulus offset (Gamlin, McDougal, Pokorny, Smith, Yau & Dacey, 2007; Kankipati, Girkin & Gamlin, 2010; Markwell, Feigl & Zele, 2010). It is unknown if intrinsic ipRGC and cone-mediated inputs to ipRGCs show circadian variation in their photon-counting activity under constant illumination. If ipRGCs demonstrate circadian variation of the pupil response under constant illumination in vivo, when in vitro ipRGC activity does not (Weng, Wong & Berson, 2009), this would support central control of the ipRGC circadian activity. A preliminary experiment was conducted to determine the spectral sensitivity of the ipRGC post-illumination pupil response under the experimental conditions, confirming the successful isolation of the ipRGC response (Gamlin, et al., 2007) for the circadian experiment. In this main experiment, we demonstrate that ipRGC photon-counting activity has a circadian rhythm under constant experimental conditions, while direct rod and cone contributions to the PLR do not. Intrinsic ipRGC contributions to the post-illumination pupil response decreased 2:46 h prior to melatonin onset for our group model, with the peak ipRGC attenuation occurring 1:25 h after melatonin onset. Our results suggest a centrally controlled evening decrease in ipRGC activity, independent of environmental light, which is temporally synchronized (demonstrates a temporal phase-advanced relationship) to the SCN mediated release of melatonin. In the future the ipRGC post-illumination pupil response could be developed as a fast, non-invasive measure of circadian rhythm. This study establishes a basis for future investigation of cortical feedback mechanisms that modulate ipRGC activity.

The post-illumination pupil response of melanopsin expressing intrinsically photosensitive retinal ganglion cells in diabetes

Purpose: This study investigates the clinical utility of the melanopsin expressing intrinsically photosensitive retinal ganglion cell (ipRGC) controlled post-illumination pupil response (PIPR) as a novel technique for documenting inner retinal function in patients with Type II diabetes without diabetic retinopathy. Methods: The post-illumination pupil response (PIPR) was measured in seven patients with Type II diabetes, normal retinal nerve fiber thickness and no diabetic retinopathy. A 488 nm and 610 nm, 7.15º diameter stimulus was presented in Maxwellian view to the right eye and the left consensual pupil light reflex was recorded. Results: The group data for the blue PIPR (488 nm) identified a trend of reduced ipRGC function in patients with diabetes with no retinopathy. The transient pupil constriction was lower on average in the diabetic group. The relationship between duration of diabetes and the blue PIPR amplitude was linear, suggesting that ipRGC function decreases with increasing diabetes duration. Conclusion: This is the first report to show that the ipRGC controlled post-illumination pupil response may have clinical applications as a non-invasive technique for determining progression of inner neuroretinal changes in patients with diabetes before they are ophthalmoscopically or anatomically evident. The lower transient pupil constriction amplitude indicates that outer retinal photoreceptor inputs to the pupil light reflex may also be affected in diabetes.

Temporal summation of intrinsically photosensitive retinal ganglion cell (ipRGC) contributions to the human pupil.

Purpose: IpRGCs mediate non-image forming functions including photoentrainment and the pupil light reflex (PLR). Temporal summation increases visual sensitivity and decreases temporal resolution for image forming vision, but the summation properties of nonimage forming vision are unknown. We investigated the temporal summation of inner (ipRGC) and outer (rod/cone) retinal inputs to the PLR. Method: The consensual PLR of the left eye was measured in six participants with normal vision using a Maxwellian view infrared pupillometer. Temporal summation was investigated using a double-pulse protocol (100 ms stimulus pairs; 0–1024 ms inter-stimulus interval, ISI) presented to the dilated fellow right eye (Tropicamide 1%). Stimulus lights (blue λmax = 460 nm; red λmax = 638 nm) biased activity to inneror outer retinal inputs to non-image forming vision. Temporal summation was measured suprathreshold (15.2 log photons.cm−2.s−1 at the cornea) and subthreshold (11.4 log photons.cm−2.s−1 at the cornea). Results: RM-ANOVAs showed the suprathreshold and subthreshold 6 second post illumination pupil response (PIPR: expressed as percentage baseline diameter) did not significantly vary for red or blue stimuli (p > .05). The PIPR for a subthreshold red 16 ms double-pulse control condition did not significantly differ with ISI (p > .05). The maximum constriction amplitude for red and blue 100 ms double- pulse stimuli did not significantly vary with ISI (p > .05). Conclusion: The non-significant changes in suprathreshold PIPR and subthreshold maximum pupil constriction indicate that inner retinal ipRGC inputs and outer retinal photoreceptor inputs to the PLR do not show temporal summation. The results suggest a fundamental difference between the temporal summation characteristics of image forming and non-image forming vision.

Melanopsin expressing intrinsically photosensitive Retinal Ganglion cells in retinal disease

Melanopsin containing intrinsically photosensitive Retinal Ganglion Cells (ipRGCs) are a class of photoreceptors with established roles in non-image forming processes. Their contributions to image forming vision may include the estimation of brightness. Animal models have been central for understanding the physiological mechanisms of ipRGC function and there is evidence of conservation of function across species. ipRGCs can be divided into 5 ganglion cell subtypes that show morphological and functional diversity. Research in humans has established that ipRGCs signal environmental irradiance to entrain the central body clock to the solar day for regulating circadian processes and sleep. In addition, ipRGCs mediate the pupil light reflex (PLR), making the PLR a readily accessible behavioural marker of ipRGC activity. Less is known about ipRGC function in retinal and optic nerve disease, with emerging research providing insight into their function in diabetes, retinitis pigmentosa, glaucoma and hereditary optic neuropathy. We briefly review the anatomical distributions, projections and basic physiological mechanisms of ipRGCs, their proposed and known functions in animals and humans with and without eye disease. We introduce a paradigm for differentiating inner and outer retinal inputs to the pupillary control pathway in retinal disease and apply this paradigm to patients with age-related macular degeneration (AMD). In these cases of patients with AMD, we provide the initial evidence that ipRGC function is altered, and that the dysfunction is more pronounced in advanced disease. Our perspective is that with refined pupillometry paradigms, the pupil light reflex can be extended to AMD assessment as a tool for the measurement of inner and outer retinal dysfunction.

The post-illumination pupil response of melanopsin expressing retinal ganglion cells

Purpose The post-illumination pupil response (PIPR) has been quantified in the literature by four metrics. The spectral sensitivity of only one metric is known and this study quantifies the other three. To optimize the measurement of the PIPR in humans, we also determine the stimulus protocol producing the largest PIPR, the duration of the PIPR, and the metric(s) with the lowest coefficient of variation. Methods The consensual pupil light reflex (PLR) was measured with a Maxwellian view pupillometer (35.6° diameter stimulus). - Experiment 1: Spectral sensitivity of four PIPR metrics [plateau, 6 s, area under curve (AUC) early and late recovery] was determined from a criterion PIPR (n = 2 participants) to a 1 s pulse at five wavelengths (409-592nm) and fitted with Vitamin A nomogram (ƛmax = 482 nm). - Experiment 2: The PLR was measured in five healthy participants [29 to 42 years (mean = 32.6 years)] as a function of three stimulus durations (1 s, 10 s, 30 s), five irradiances spanning low to high melanopsin excitation levels (retinal irradiance: 9.8 to 14.8 log quanta.cm-2.s-1), and two wavelengths, one with high (465 nm) and one with low (637 nm) melanopsin excitation. Intra and inter-individual coefficients of variation (CV) were calculated. Results The melanopsin (opn4) photopigment nomogram adequately described the spectral sensitivity derived from all four PIPR metrics. The largest PIPR amplitude was observed with 1 s short wavelength pulses (retinal irradiance ≥ 12.8 log quanta.cm-2.s-1). Of the 4 PIPR metrics, the plateau and 6 s PIPR showed the least intra and inter-individual CV (≤ 0.2). The maximum duration of the sustained PIPR was 83.4 ± 48.0 s (mean ± SD) for 1 s pulses and 180.1 ± 106.2 s for 30 s pulses (465 nm; 14.8 log quanta.cm-2.s-1). Conclusions All current PIPR metrics provide a direct measure of intrinsic melanopsin retinal ganglion cell function. To measure progressive changes in melanopsin function in disease, we recommend that the intrinsic melanopsin response should be measured using a 1 s pulse with high melanopsin excitation and the PIPR should be analyzed with the plateau and/or 6 s metrics. That the PIPR can have a sustained constriction for as long as 3 minutes, our PIPR duration data provide a baseline for the selection of inter-stimulus intervals between consecutive pupil testing sequences.

Melanopsin ganglion cell function in early age-related macular degeneration

Purpose Melanopsin-expressing retinal ganglion cells (mRGCs) have non-image forming functions including mediation of the pupil light reflex (PLR). There is limited knowledge about mRGC function in retinal disease. Initial retinal changes in age-related macular degeneration (AMD) occur in the paracentral region where mRGCs have their highest density, making them vulnerable during disease onset. In this cross-sectional clinical study, we measured the PLR to determine if mRGC function is altered in early stages of macular degeneration. Methods Pupil responses were measured in 8 early AMD patients (AREDS 2001 classification; mean age 72.6 ± 7.2 years, 5M, and 3F) and 12 healthy control participants (mean age 66.6 ± 6.1 years, 8M and 4F) using a custom-built Maxwellian-view pupillometer. Stimuli were 0.5 Hz sinewaves (10 s duration, 35.6° diameter) of short wavelength light (464nm, blue; retinal irradiance = 14.5 log quanta.cm-2.s-1) to produce high melanopsin excitation and of long wavelength light (638nm, red; retinal irradiance = 14.9 log quanta.cm-2.s-1), to bias activation to outer retina and provide a control. Baseline pupil diameter was determined during a 10 s pre-stimulus period. The post illumination pupil response (PIPR) was recorded for 40 s. The 6 s PIPR and maximum pupil constriction were expressed as percentage baseline (M ± SD). Results The blue PIPR was significantly less sustained (p<0.01) in the early AMD group (75.49 ± 7.88%) than the control group (58.28 ± 9.05%). The red PIPR was not significantly different (p>0.05) between the early AMD (84.79 ± 4.03%) and control groups (82.01 ± 5.86%). Maximum constriction amplitude in the early AMD group for blue (43.67 ± 6.35%) and red (48.64 ± 6.49%) stimuli were not significantly different to the control group for blue (39.94 ± 3.66%) and red (44.98 ± 3.15%) stimuli (p>0.05). Conclusions These results are suggestive of inner retinal mRGC deficits in early AMD. This non-invasive, objective measure of pupil responses may provide a new method for quantifying mRGC function and monitoring AMD progression.

Effect of the pyrethroid insecticide fenvalerate on sodium current of rat dorsal root ganglion neuron

Fenvalerate is a widely used pyrethroid insecticide. The report presents our findings on the effect of fenvalerate on isolated whole-cell sodium currents in single rat dorsal root ganglionic neurons in culture, studied with patch-clamp technique. Fenvalerate decreased the amplitude of whole-cell sodium current and slowed the inactivation and tail current kinetics.

Influence of Extracellular Matrix Components on the Expression of Integrins and Regeneration of Adult Retinal Ganglion Cells

Purpose Retinal ganglion cells (RGCs) are exposed to injury in a variety of optic nerve diseases including glaucoma. However, not all cells respond in the same way to damage and the capacity of individual RGCs to survive or regenerate is variable. In order to elucidate factors that may be important for RGC survival and regeneration we have focussed on the extracellular matrix (ECM) and RGC integrin expression. Our specific questions were: (1) Do adult RGCs express particular sets of integrins in vitro and in vivo? (2) Can the nature of the ECM influence the expression of different integrins? (3) Can the nature of the ECM affect the survival of the cells and the length or branching complexity of their neurites? Methods Primary RGC cultures from adult rat retina were placed on glass coverslips treated with different substrates: Poly-L-Lysine (PL), or PL plus laminin (L), collagen I (CI), collagen IV (CIV) or fibronectin (F). After 10 days in culture, we performed double immunostaining with an antibody against beta III-Tubulin to identify the RGCs, and antibodies against the integrin subunits: alpha V, alpha 1, alpha 3, alpha 5, beta 1 or beta 3. The number of adhering and surviving cells, the number and length of the neurites and the expression of the integrin subunits on the different substrates were analysed. Results PL and L were associated with the greatest survival of RGCs while CI provided the least favourable conditions. The type of substrate affected the number and length of neurites. L stimulated the longest growth. We found at least three different types of RGCs in terms of their capacity to regenerate and extend neurites. The different combinations of integrins expressed by the cells growing on different substrata suggest that RGCs expressed predominantly alpha 1 beta 1 or alpha 3 beta 1 on L, alpha 1 beta 1 on CI and CIV, and alpha 5 beta 3 on F. The activity of the integrins was demonstrated by the phosphorylation of focal adhesion kinase (FAK). Conclusions Adult rat RGCs can survive and grow in the presence of different ECM tested. Further studies should be done to elucidate the different molecular characteristics of the RGCs subtypes in order to understand the possible different sensitivity of different RGCs to damage in diseases like glaucoma in which not all RGCs die at the same time.

Adult rat retinal ganglion cells and glia can be printed by piezoelectric inkjet printing.

We have investigated whether inkjet printing technology can be extended to print cells of the adult rat central nervous system (CNS), retinal ganglion cells (RGC) and glia, and the effects on survival and growth of these cells in culture, which is an important step in the development of tissue grafts for regenerative medicine, and may aid in the cure of blindness. We observed that RGC and glia can be successfully printed using a piezoelectric printer. Whilst inkjet printing reduced the cell population due to sedimentation within the printing system, imaging of the printhead nozzle, which is the area where the cells experience the greatest shear stress and rate, confirmed that there was no evidence of destruction or even significant distortion of the cells during jet ejection and drop formation. Importantly, the viability of the cells was not affected by the printing process. When we cultured the same number of printed and non-printed RGC/glial cells, there was no significant difference in cell survival and RGC neurite outgrowth. In addition, use of a glial substrate significantly increased RGC neurite outgrowth, and this effect was retained when the cells had been printed. In conclusion, printing of RGC and glia using a piezoelectric printhead does not adversely affect viability and survival/growth of the cells in culture. Importantly, printed glial cells retain their growth-promoting properties when used as a substrate, opening new avenues for printed CNS grafts in regenerative medicine.