A modified silent substitution electroretinography protocol to separate photoreceptor subclass function in lightly sedated dogs.

OBJECTIVE: A previously published study successfully isolated photoreceptor responses from canine rods, long/medium-wavelength (L/M) cones, and short-wavelength (S) cones using silent substitution electroretinography (ERG) performed under general anesthesia. We hypothesized that responses would be similar in dogs under sedation and that a curtailed protocol suitable for use in clinical patients could effectively isolate responses from all three photoreceptor subtypes. ANIMALS STUDIED: Three normal adult purpose-bred beagles (2 females and 1 male). METHODS: Dogs were dark-adapted for 1 hour. Sine wave color stimuli were delivered using LEDs in a Ganzfeld dome. The ERG protocol under anesthesia was performed as previously published; sedated ERG protocols were performed after a 3-day washout period. Intravenous sedation (dexmedetomidine 1.25 mcg/kg, butorphanol 0.1 mg/kg) was administered for sedation. Statistical analysis was performed using two-way repeated-measures ANOVA and linear regression. RESULTS: In both anesthetized and sedated animals, rod-derived responses peaked at low frequency (4-12 Hz), L/M-cone responses peaked at high frequency (32-38 Hz), and S-cone responses peaked at low frequency (4-12 Hz). The frequencies eliciting maximal responses were similar in sedated and anesthetized protocols, although rod amplitudes were significantly higher in the sedated protocols compared with anesthetized (P < .001). CONCLUSION: We present a clinically applicable method to consistently isolate rod and cone subclass function in sedated dogs. This may allow detailed evaluation of photoreceptor function in clinical patients with rod or cone subclass deficits without the need for general anesthesia or protracted adaptation times.

Impact of network topology on the stability of DC microgrids.

We probe the stability of Watts-Strogatz DC microgrids, in which droop-controlled producers and constant power load consumers are homogeneously distributed and obey Kirchhoff's circuit laws. The concept of survivability is employed to evaluate the system's response to Dirac-delta voltage perturbations at single nodes. A fixed point analysis of the power grid model yields that there is only one relevant attractor. Using a set of simulations with random networks, we investigate correlations between survivability and three topological network measures: the share of producers in the network and the degree and the average neighbor degree of the perturbed node. Depending on the imposed voltage and current limits, the stability is optimized for low node degrees or a specific share of producers. Based on our findings, we provide an insight into the local dynamics of the perturbed system and derive explicit guidelines for the design of resilient DC power grids.

Human S-cone electroretinograms obtained by silent substitution stimulation.

We used triple silent substitution stimuli to characterize human S-cone electroretinograms (ERGs) in normal trichromats. Short-wavelength-cone (S-cone) ERGs were found to have different morphological features and temporal frequency response characteristics compared to ERGs derived from L-cones, M-cones, and rod photoreceptors in normal participants. Furthermore, in two cases of retinal pathology, blue cone monochromatism (BCM) and enhanced S-cone syndrome (ESCS), S-cone ERGs elicited by our stimuli were preserved and enhanced, respectively. The results from both normal and pathological retinae demonstrate that triple silent substitution stimuli can be used to generate ERGs that provide an assay of human S-cone function.

The morphology of human rod ERGs obtained by silent substitution stimulation.

PURPOSE: To record transient ERGs from the light-adapted human retina using silent substitution stimuli which selectively reflect the activity of rod photoreceptors. We aim to describe the morphology of these waveforms and examine how they are affected by the use of less selective stimuli and by retinal pathology. METHODS: Rod-isolating stimuli with square-wave temporal profiles (250/250 ms onset/offset) were presented using a 4 primary LED ganzfeld stimulator. Experiment 1: ERGs were recorded using a rod-isolating stimulus (63 ph Td, rod contrast, C rod = 0.25) from a group (n = 20) of normal trichromatic observers. Experiment 2: Rod ERGs were recorded from a group (n = 5) using a rod-isolating stimulus (C rod = 0.25) which varied in retinal illuminance from 40 to 10,000 ph Td. Experiment 3: ERGs were elicited using 2 kinds of non-isolating stimuli; (1) broadband and (2) rod-isolating stimuli which contained varying degrees of L- and M-cone excitation. Experiment 4: Rod ERGs were recorded from two patient groups with rod monochromacy (n = 3) and CSNB (type 1; n = 2). RESULTS: The rod-isolated ERGs elicited from normal subjects had a waveform with a positive onset component followed by a negative offset. Response amplitude was maximal at retinal illuminances <100 ph Td and was virtually abolished at 400 ph Td. The use of non-selective stimuli altered the ERG waveform eliciting more photopic-like ERG responses. Rod ERGs recorded from rod monochromats had similar features to those recorded from normal trichromats, in contrast to those recorded from participants with CSNB which had an electronegative appearance. CONCLUSIONS: Our results demonstrate that ERGs elicited by silent substitution stimuli can selectively reflect the operation of rod photoreceptors in the normal, light-adapted human retina.

[Recovery time as a potential new progression parameter for patients with advanced glaucomatous optic atrophy]. / Erholungszeit als potenzieller neuer Verlaufsparameter für Patienten mit fortgeschrittener glaukomatöser Optikusatrophie.

BACKGROUND: Functional tests in glaucoma diagnosis can monitor a potential perimetric progression. However white-on-white perimetry is limited in advanced glaucoma, whereas contrasts were detected reproducibly. Especially when stressing (i. e. adapting) the visual system, subsequent measurements yielded different results-the visual system needs a "recovery time". In the present study the recovery time was investigated in patients with advanced glaucoma. Additionally correlation analysis was done with standard perimetric parameters. MATERIAL AND METHODS: Temporal contrast sensitivity (TCS) and recovery time (RT) were measured using the Erlanger Flicker Test in 61 probands (15 normals, 25 primary open-angle glaucomas, 17 secondary open-angle glaucomas, 4 narrow-angle glaucoma). Additionally, ophthalmological examinations and perimetry (Octopus G1) was done. RESULTS: (1) TCS was significantly reduced in patients with advanced glaucoma (p < 0.001). (2) Test stimuli with 3 and 5 % contrast showed a large variability in contrast to higher contrasts (12 %, 25 %, 35 %). (3) RT12%, RT25% and RT35% were significantly prolonged in advanced glaucoma (p < 0.001). (4) RT25% correlated significantly with mean defect (p = 0.015). CONCLUSION: Recovery time seems to be a potential parameter in advanced glaucoma follow-up, as it is reproducible, independently of cataract and fixation.

Objective visual field determination in forensic ophthalmology with an optimized 4-channel multifocal VEP perimetry system: a case report of a patient with retinitis pigmentosa.

We present the case of a 59-year-old male patient with progressive vision impairment and consecutive visual field narrowing ("tunnel view") for 7 years and a known retinitis pigmentosa for 5 years. The remaining Goldmann perimetric visual field at time reported was less than 5°. A request for blindness-related social benefits was rejected because an ophthalmologic expert assessment suggested malingering. This prompted us to assess an objective determination of the visual field using multifocal VEPs. Objective visual field recordings were performed with a four-channel multifocal VEP-perimeter using 58 stimulus fields (pattern reversal dartboard stimulus configuration). The correlated signal data were processed using an off-line method. At each field, the recording from the channel with the maximal signal-to-noise ratio (SNR) was retained, thus resulting in an SNR optimized virtual recording. Analysis of VEP signals was performed for each single field and concentric rings and compared to an average response measured in five healthy subjects. Substantial VEP responses could be identified in three fields within the innermost ring (eccentricity, 1.7°) for both eyes, although SNR was generally low. More eccentric stimuli did not elicit reliable VEP responses. The mfVEP recording was correlated with perimetric visual field data. The current SNR optimization by using the channel with the largest SNR provides a good method to extract useful data from recordings and may be appropriate for the use in forensic ophthalmology.

Retinal disorders in northern Brazilian patients treated with chloroquine assessed by multifocal ERG.

The effects of chloroquine intake on the retinal function in a Brazilian population of patients were assessed by multifocal electroretinography. Twenty-four randomly chosen eyes of patients treated with chloroquine for rheumatoid arthritis and systemic lupus erythematosus were examined using multifocal electroretinography (mfERG). Control measurements were acquired from 21 randomly chosen eyes of age-matched healthy subjects. None of the study participants had an inherited retinal disease or a Snellen visual acuity reduced to less than 20/40. In patients and control subjects, cumulative chloroquine dose, total daily dose, duration of treatment, retinal examination, visual field defects, visual acuity, and the mfERG were assessed. The average amplitudes and implicit times of the N1, P1, and P2 components of the mfERGs were measured in the central hexagon (R1) and in five rings (R2-R6). The values measured in patients and normal subjects were compared. The P1 amplitudes in R2 were significantly decreased in the patients. In addition, the amplitudes of N1 and N2 in R1 were significantly smaller in the patients. The implicit times of none of the components were significantly different between patients and controls. The response amplitude was not significantly correlated with cumulative dose and duration of intake. There was no correlation with retinal appearance, visual field, and visual acuity. In agreement with earlier data, the central mfERG amplitudes were decreased in chloroquine patients indicating functional alterations in the retina. These changes are also present in a Brazilian population suggesting that the effects of chloroquine are general and that genetic background and life circumstances probably have, if at all, only little effect.

L- and M-cone input to 12Hz and 30Hz flicker ERGs across the human retina.

We recorded L- and M-cone isolating ERGs from human subjects using a silent substitution technique at temporal rates of 12 and 30 Hz. These frequencies isolate the activity of cone-opponent and non-opponent post-receptoral mechanisms, respectively. ERGs were obtained using a sequence of stimuli with different spatial configurations comprising; (1) circular stimuli of different sizes which increased in 10° steps up to 70°diameter, or (2) annular stimuli with a 70° outer diameter but with different sized central ablations from 10° up to 60°. L- and M-cone isolating ERGs were obtained from five colour normal subjects using a DTL fibre electrode. Fourier analysis of the ERGs was performed and we measured the amplitude of the first harmonic of the response. For 12 Hz ERGs the L:M cone response amplitude ratio (L:M(ERG)) was close to unity and remained stable irrespective of the spatial configuration of the stimulus. The maintenance of this balanced ratio points to the existence of cone selective input across the human retina for the L-M cone opponent mechanism. For 30 Hz the L:M(ERG) ratio was greater than unity but varied depending upon which region of the retina was being stimulated. This variation we consider to be a consequence of the global response properties of M-cone ERGs rather than representing a real variation in L:M cone ratios across the retina.

L- and M-cone isolating ERGs: LED versus CRT stimulation.

Using double silent substitution, it is possible to generate L-cone and M-cone isolating electroretinograms (ERGs) on a CRT. A major limitation of the technique is that the depth of modulation of cone classes is limited by the restricted luminance of the phosphors and their spectral overlap. To address this problem we have ported the technique to a four-color LED Ganzfeld stimulus (Diagnosis ColorDome). This allows higher retinal illuminances, higher contrasts, and triple silent substitution. With careful control over the retinal area stimulated, we show that the same data can be recorded from both CRT and LED stimuli when luminance, size and cone contrast are kept constant. Importantly, the different temporal profiles of the two devices do not influence the ERG amplitude and phase plots. We present data over a much wider range of luminances (up to about 10,000 trolands) and contrasts with the LED stimulator than previously reported with CRT screens. We conclude that the close resemblance between data obtained with an LED stimulator and with a CRT screen indicate that the differences have a purely physiological origin.

Dendritic thickness: a morphometric parameter to classify mouse retinal ganglion cells

To study the dendritic morphology of retinal ganglion cells in wild-type mice we intracellularly injected these cells with Lucifer yellow in an in vitro preparation of the retina. Subsequently, quantified values of dendritic thickness, number of branching points and level of stratification of 73 Lucifer yellow-filled ganglion cells were analyzed by statistical methods, resulting in a classification into 9 groups. The variables dendritic thickness, number of branching points per cell and level of stratification were independent of each other. Number of branching points and level of stratification were independent of eccentricity, whereas dendritic thickness was positively dependent (r = 0.37) on it. The frequency distribution of dendritic thickness tended to be multimodal, indicating the presence of at least two cell populations composed of neurons with dendritic diameters either smaller or larger than 1.8 æm ("thin" or "thick" dendrites, respectively). Three cells (4.5 percent) were bistratified, having thick dendrites, and the others (95.5 percent) were monostratified. Using k-means cluster analysis, monostratified cells with either thin or thick dendrites were further subdivided according to level of stratification and number of branching points: cells with thin dendrites were divided into 2 groups with outer stratification (0-40 percent) and 2 groups with inner (50-100 percent) stratification, whereas cells with thick dendrites were divided into one group with outer and 3 groups with inner stratification. We postulate, that one group of cells with thin dendrites resembles cat ß-cells, whereas one group of cells with thick dendrites includes cells that resemble cat a-cells.

Dendritic thickness: a morphometric parameter to classify mouse retinal ganglion cells.

To study the dendritic morphology of retinal ganglion cells in wild-type mice we intracellularly injected these cells with Lucifer yellow in an in vitro preparation of the retina. Subsequently, quantified values of dendritic thickness, number of branching points and level of stratification of 73 Lucifer yellow-filled ganglion cells were analyzed by statistical methods, resulting in a classification into 9 groups. The variables dendritic thickness, number of branching points per cell and level of stratification were independent of each other. Number of branching points and level of stratification were independent of eccentricity, whereas dendritic thickness was positively dependent (r = 0.37) on it. The frequency distribution of dendritic thickness tended to be multimodal, indicating the presence of at least two cell populations composed of neurons with dendritic diameters either smaller or larger than 1.8 microm ("thin" or "thick" dendrites, respectively). Three cells (4.5%) were bistratified, having thick dendrites, and the others (95.5%) were monostratified. Using k-means cluster analysis, monostratified cells with either thin or thick dendrites were further subdivided according to level of stratification and number of branching points: cells with thin dendrites were divided into 2 groups with outer stratification (0-40%) and 2 groups with inner (50-100%) stratification, whereas cells with thick dendrites were divided into one group with outer and 3 groups with inner stratification. We postulate, that one group of cells with thin dendrites resembles cat beta-cells, whereas one group of cells with thick dendrites includes cells that resemble cat alpha-cells.

Spatial receptive field properties of lateral geniculate cells in the owl monkey (Aotus azarae) at different contrasts: a comparative study.

Several physiological properties of owl monkey lateral geniculate nucleus (LGN) cells were studied to verify whether its nocturnal habit has an influence on the organization of its subcortical visual system. Receptive field (RF) dimensions were measured using drifting gratings and bipartite field stimuli. We found that owl monkey cells LGN have larger RFs and were on average more non-linear than those of diurnal monkeys. But, as in other anthropoids, there is an increase in RF centre size with increasing eccentricity, and there is a limited correlation between these centre sizes and retinal ganglion cell dendritic tree sizes. The influence of contrast on sizes and peak sensitivities of RF centres and surrounds and on the response phases was studied. Both the sizes and peak sensitivities of the RF centres and surrounds decrease as contrast increases. As a result, the responses to low spatial frequency stimuli saturate with increasing contrast. Estimates of contrasts at half-maximal responses confirm the presence of saturation. It was found that the magnocellular cells saturate more strongly than parvocellular cells. The response phase increases with increasing contrast. These data resemble those obtained in the common marmoset, indicating that these are basic features of the primate visual system. We conclude that during evolution and as an adaptation to a nocturnal lifestyle, cells in the owl monkey LGN display an increased spatial integration in comparison with diurnal primate species, without a change in the basic organization common to the primate subcortical visual system.

Photoreceptor topography and cone-specific electroretinograms.

It is implicit in many cone-specific ERG studies that the amplitude is proportional to the numbers of cones stimulated. The objective of these experiments was to test this idea by comparing ERGs obtained from different areas of the retina with histological data on cone-density distributions. The histology (Curcio et al., 1990) shows that the cumulative number of cones in the human retina increases exponentially with stimulus diameter between 0- and 40-deg eccentricity. L-, M-, and (L+M) cone-driven 30-Hz ERGs were obtained from a series of stimuli with one of the following configurations: (1) Circular stimuli of different angular subtense up to 70-deg diameter. (2) Annuli with 70-deg outer diameter but variable inner diameter. (3) Annuli of constant area but increasing eccentricity. Cone contrasts were equalized for each stimulus condition. The modulated and nonmodulated regions of the screen had the same mean hue and luminance. The data suggest that the L+M cone ERG amplitude increases with stimulus diameter in direct proportion to the estimated number of cones stimulated. Furthermore, the total L+M responses appear to be predicted from individual L and M responses by simple linear summation for both the disc and annular stimuli.

M-cone opsin gene number does not correlate with variation in L/M-cone sensitivity.

Molecular genetic studies demonstrate that the human cone opsin gene array on the q-arm of the X-chromosome typically consists of one long-wave-sensitive (L) cone opsin gene and from one to several middle-wave-sensitive (M) cone opsin genes. Although the presence of the single L-cone opsin gene and at least one M-cone opsin gene is essential for normal red-green colour discrimination, the function of the additional M-cone opsin genes is still unclear. To investigate whether any variations in phenotype correlate with differences in the number of M-cone opsin genes, we selected 13 normal trichromat males, for whom four independent molecular techniques have exactly determined their number of M-cone opsin genes, ranging from one to four. Their phenotype was characterized by estimating their foveal L- to M-cone ratio from heterochromatic flicker photometric (HFP) thresholds, by measuring the wavelength corresponding to their 'unique yellow', and by determining their L- and M-cone modulation thresholds (CMTs). No correlation was found between these psychophysical measures and the number of M-cone opsin genes. Although, we found a reasonably good correlation between the L/M-cone ratios based on HFP and on CMT, we did not find any correlation between the estimated L/M-cone ratios and the settings of 'unique yellow'. Our results accord with previous molecular genetic studies that suggest that only the first two genes in the X-linked opsin gene array are expressed.

Rod-/L-cone and rod-/M-cone interactions in electroretinograms at different temporal frequencies.

We recorded electroretinograms (ERGs) under stimulus conditions that only modulated one cone type: either the L- or the M-cones. In these conditions the rods were also modulated. We measured the ERG responses at different temporal frequencies. A simple model that assumes that the first harmonic components of the responses are the result of a vector addition of rod- and cone-driven ERG responses can explain the data satisfactorily for temporal frequencies at and above 6 Hz. From fits of the model to the data, estimates of the gains and phases of the rod- and cone-driven responses can be obtained. At 6 Hz, the fundamental responses originate exclusively in the rods. The gains of the cone-driven responses are substantial at the other temporal frequencies, are maximal at 12 or 18 Hz, and then decrease with increasing temporal frequencies. The gains of the rod-driven responses decrease more steeply with increasing temporal frequencies than the cone gains. Furthermore, the rod and cone phases decrease approximately linearly with temporal frequency, suggesting that they are mainly determined by a response delay. The response delay in the rod-driven ERGs is larger than in the cone-driven ERGs.

Macular dystrophy with protan genotype and phenotype studied with cone type specific ERGs.

PURPOSE: To determine the L- and M-cone driven ERG responses in a male patient with macular dystrophy and a protan phenotype. METHODS: We measured large field ERG thresholds to stimuli which modulated exclusively the L- or the M-cones or the two in various combinations (both in-phase and in counterphase). In none of the stimuli, the S-cones were modulated. Additionally, standard and multifocal ERGs were measured. Analysis of the L- and M-cone pigment genes was performed by means of PCR, RFLP analysis and DNA sequencing techniques. RESULTS: Macular dystrophy was revealed by the markedly abnormal multifocal ERGs in presence of near normal standard ERGs. The large field ERG responses were exclusively driven by the M-cones with enlarged thresholds when compared with otherwise normal protanopes. In addition, the M-cone driven ERG response phases were abnormal. Pigment gene analysis confirmed a protan genotype with the presence of a single 5'red/3'green hybrid pigment gene. CONCLUSIONS: Our novel stimulus technique allows a reliable analysis of the separate cone pathways even in cases with macular dysfunction. The increased thresholds and the abnormal phase behavior of the M-cone driven ERGs reflect altered mechanisms of the retinal physiology in this patient. The data strongly suggest that the macular dystrophy and the protanopia have independent origins.

L- and M-cone-driven electroretinograms in Stargardt's macular dystrophy-fundus flavimaculatus.

PURPOSE: To study the dynamics of the long (L)- and middle (M)-wavelength-sensitive cone-driven pathways and their interactions in patients with Stargardt's macular dystrophy-fundus flavimaculatus (SMD-FF) and to correlate them with other clinical parameters and individual genotypes. METHODS: Forty-seven patients with SMD-FF participated in the study. In addition to standard 30-Hz flicker electroretinograms (30-Hz fERG), ERG responses were measured to stimuli that modulated exclusively the L or the M cones (L/M cones) or the two simultaneously. Blood samples were screened for mutations in the 50 exons of the ABCA4 gene. RESULTS: Patients with SMD-FF did not show a decrease in the mean L/M-cone-driven ERG sensitivity, but there was a significant increase in the interindividual variability. The mean L-/M-cone weighting ratio was normal. However, the L-cone-driven ERG was significantly phase delayed, whereas the M-cone-driven ERG was significantly phase advanced. These phase changes were significantly correlated with disease duration. The amplitude and implicit time of the standard 30-Hz fERG both correlated significantly with the L/M-cone-driven ERG sensitivity and with the phase difference between the L/M-cone-driven ERGs, indicating the complex origin of the standard 30-Hz fERG. Probable disease-associated mutations in the ABCA4 gene were found in 40 of 45 patients, suggesting that they form a genetically fairly uniform SMD-FF study group. There was no correlation between the genotype and the L/M-cone-driven ERGS: CONCLUSIONS: The changes in L/M-cone-driven ERG sensitivity and phase possibly represent two independent disease processes. The phase changes are similar to those found in patients with retinitis pigmentosa and possibly are a general feature of retinal dystrophies.

Influence of contrast on the responses of marmoset lateral geniculate cells to drifting gratings.

The responses of lateral geniculate nucleus (LGN) cells in the common marmoset (Callithrix jacchus) to drifting luminance or cone isolating gratings of different spatial frequencies and contrasts were measured. The response noise, defined as the variability of the responses to single sweeps in the complex plane, was independent of stimulus contrast and spatial frequency but increased with increasing overall responsiveness of the cell. The signal-to-noise ratio of parvocellular (PC) cells was smaller than of magnocellular (MC) cells. At each contrast, the response amplitude as a function of spatial frequency could be described with a difference of Gaussians model. With this model, the sizes and the peak sensitivities of the receptive field centers and surrounds were estimated. It was found that receptive field center and surround sizes of LGN cells decrease slightly with increasing contrast. Further, the peak sensitivity decreases with increasing contrast. The two factors are involved in a decrease in responsivity (the response per unit contrast) with increasing contrast which is compatible to response saturation for low spatial frequency stimuli. PC cells did not saturate as much to luminance stimuli although some saturation was found with cone isolating gratings. We found that the response phase lag of both PC and MC cells decreased with increasing contrast, which cannot be explained on the basis of linear response behavior. Apparently the phase of LGN cell responses to drifting gratings is altered in comparison with the retinal inputs by additional nonlinearities.

Large phase differences between L-cone- and M-cone-driven electroretinograms in retinitis pigmentosa.

PURPOSE: To study the dynamics and interactions of the signals originating in the long- (L-) and middle (M)-wavelength-sensitive cone pathways in patients with retinitis pigmentosa (RP). METHODS: Twenty-six patients with RP and 29 normal subjects participated in the study. Electroretinographic (ERG) responses were measured to stimuli that modulated exclusively the L- or the M-cones or the two simultaneously (both in-phase and in counter-phase) with varying ratios of L- to M-cone contrasts. S-cones were not modulated. RESULTS: The data of the normal subjects and of the patients can be described by a model in which the amplitudes and the phases of the signals originating in the L- and M-cones are vector summed. In the RP patients, there was a general reduction in ERG sensitivity. The L-cone-driven ERG response was significantly delayed, whereas the M-cone-driven ERG response was phase advanced. CONCLUSIONS: Large dynamic differences between L- and M-cone-driven ERGs can be detected in RP. As a result, the interaction between the L- and M-cone systems, when modulated simultaneously at 30 Hz, is subtractive in RP patients and additive in normal subjects. Our data show that the use of only a standard white flicker ERG might lead to a misinterpretation of the mechanisms involved in retinal disorders, because the phases of different cone-driven responses are not considered.

L- and M-cone driven ERGs are differently altered in Best's macular dystrophy.

To study the L- and M-cone pathways and their interactions in patients with Best's macular dystrophy (BMD), ERG response thresholds were measured to stimuli which modulated exclusively the L- or the M-cones, or both in various combinations. The ERG threshold data could be described with a vector addition model. Compared with normals, BMD patients showed generally larger amplitudes of the L-cone driven ERGs. However, the M-cone driven ERGs were similar in amplitude but significantly phase advanced. The data confirm our previous observations that L- and M-cone pathways can be affected differently by retinal degeneration, despite their large physiological and biochemical similarities.