Evolutionary and homeostatic changes in morphology of visual dendrites of Mauthner cells in Astyanax blind cavefish.

Mauthner cells are the largest neurons in the hindbrain of teleost fish and most amphibians. Each cell has two major dendrites thought to receive segregated streams of sensory input: the lateral dendrite receives mechanosensory input while the ventral dendrite receives visual input. These inputs, which mediate escape responses to sudden stimuli, may be modulated by the availability of sensory information to the animal. To understand the impact of the absence of visual information on the morphologies of Mauthner cells during developmental and evolutionary time scales, we examined the teleost Astyanax mexicanus. This species of tetra is found in two morphs: a seeing surface fish and a blind cavefish. We compared the structure of Mauthner cells in surface fish raised under daily light conditions, in surface fish raised in constant darkness, and in two independent lineages of cave populations. The length of ventral dendrites of Mauthner cells in dark-raised surface fish larvae were longer and more branched, while in both cave morphs the ventral dendrites were smaller or absent. The absence of visual input in surface fish with normal eye development leads to a homeostatic increase in dendrite size, whereas over evolution, the absence of light led to the loss of eyes and a reduction in dendrite size.

Spooky Interaction at a Distance in Cave and Surface Dwelling Electric Fishes.

Glass knifefish (Eigenmannia) are a group of weakly electric fishes found throughout the Amazon basin. Their electric organ discharges (EODs) are energetically costly adaptations used in social communication and for localizing conspecifics and other objects including prey at night and in turbid water. Interestingly, a troglobitic population of blind cavefish Eigenmannia vicentespelea survives in complete darkness in a cave system in central Brazil. We examined the effects of troglobitic conditions, which includes a complete loss of visual cues and potentially reduced food sources, by comparing the behavior and movement of freely behaving cavefish to a nearby epigean (surface) population (Eigenmannia trilineata). We found that the strengths of electric discharges in cavefish were greater than in surface fish, which may result from increased reliance on electrosensory perception, larger size, and sufficient food resources. Surface fish were recorded while feeding at night and did not show evidence of territoriality, whereas cavefish appeared to maintain territories. Surprisingly, we routinely found both surface and cavefish with sustained differences in EOD frequencies that were below 10 Hz despite being within close proximity of about 50 cm. A half century of analysis of electrosocial interactions in laboratory tanks suggest that these small differences in EOD frequencies should have triggered the "jamming avoidance response," a behavior in which fish change their EOD frequencies to increase the difference between individuals. Pairs of fish also showed significant interactions between EOD frequencies and relative movements at large distances, over 1.5 m, and at high differences in frequencies, often >50 Hz. These interactions are likely "envelope" responses in which fish alter their EOD frequency in relation to higher order features, specifically changes in the depth of modulation, of electrosocial signals.

Morphological malleability of the lateral line allows for surface fish (Astyanax mexicanus) adaptation to cave environments.

The lateral line is the primary modality fish use to create a hydrodynamic image of their environment. These images contribute to a variety of behaviors, from rheotaxis to escape responses. Here we discern the contributions of visual and lateral line modalities in hunting behavior of larvae that have developed under different photic conditions. In particular, cave animals have a hypertrophied sense of mechanosensation, and we studied the common animal model cavefish Astyanax mexicanus and its closest related surface relative. We raised larvae in a diurnal light-dark regimen and in complete darkness. We then examined the distribution of neuromasts in their lateral lines, and their hunting performance in light and dark conditions, with and without the contribution of the lateral line. We report that all larva depend on the lateral line for success in hunting and that surface fish raised in the dark have a greater dependency on the lateral line.

Differences in behavior between surface and cave Astyanax mexicanus may be mediated by changes in catecholamine signaling.

Astyanax mexicanus is a teleost fish that is in the process of allopatric speciation. Ancestral Astyanax are found in surface rivers and derived blind forms are found in cave systems. Adaptation to life in nutrient poor caves without predation includes the evolution of enhanced food seeking behaviors and loss of defensive responses. These behavioral adaptations may be mediated by changes in catecholaminergic control systems in the brain. We examined the distribution of tyrosine hydroxylase, a conserved precursor for the synthesis of the catecholamines dopamine and noradrenaline, in the brains of surface and cave Astyanax using immunohistochemistry. We found differences in tyrosine hydroxylase staining in regions that are associated with nonvisual sensory perception, motor control, endocrine release, and attention. These differences included significant increases in the diameters of tyrosine hydroxylase immunoreactive soma in cave Astyanax in the olfactory bulb, basal telencephalon, preoptic nuclei, ventral thalamus, posterior tuberculum, and locus coeruleus. These increases in modulation by dopamine and noradrenaline likely indicate changes in behavioral control that underlie adaptations to the cave environment.

Extreme Adaptation in Caves.

Cave adaptation leads to unique anatomical specializations in many taxonomic groups. As the role of vision is reduced or disappears in a subterranean environment, other specializations arise to allow the organism to successfully detect and interact with their environment. A suite of unique, convergent phenotypes associated with subterranean adaptation has emerged (termed troglomorphy), with reduction or loss of pigmentation and eyes being the most conspicuous. Two vertebrate groups that have successfully colonized and adapted to subterranean environments are cavefishes and cave salamanders. There are many shared troglomorphic anatomical characters shared between these two groups, and we describe herein the morphological traits that are unique to fishes and salamanders that are adapted to caves and other subterranean habitats. Troglobionts, animals strictly bound and adapted to underground habitats, are outcomes of not just regressive evolution, but also constructive adaptation. There are skeletal changes, such as broadening and flattening of the head, as well as hypertrophy of non-visual modalities. Cavefishes and salamanders have lost eyes and pigmentation, but also enhanced mechanosenzation, chemosenzation and, in some cases, electroreception. Both cavefishes and cave salamanders have become important models in the study of the ecology, behavior, and evolution of subterranean colonization and adaptation. However, our knowledge is primarily limited to a few taxa and many questions remain to be studied. Anat Rec, 2018. © 2018 American Association for Anatomy.

Retinal morphology in Astyanax mexicanus during eye degeneration.

The teleost Astyanax mexicanus is one species extant in two readily available forms. One that lives in Mexican rivers and various convergent forms that live in nearby caves. These fish are born with eyes but in the cavefish, they degenerate during development. It is known that the lens of cavefish undergoes apoptosis and that some cells in the neuroretina also die. It has not been described, however, if glia and various components of the neuroretina form before complete eye degeneration. Here we examined the development of the retina of the closest living ancestor that lives in the rivers and two independently adapted of cavefish. We report that although the neuroretina is smaller and more compact, it has all cell types and layers including amacrine cells and Müller glia. While various makers for photoreceptors are present in the cavefish inner segments, the outer segments of the photoreceptors in cavefish are missing from the earliest stages examined. This shows that the machinery for visual transducing discs might still be present but not organized in one part of the cell. It is interesting to note that the deficiencies in Astyanax cavefish resemble retinal diseases, such as retinitis pigmentosa.

Sensorimotor Transformations in the Zebrafish Auditory System.

Organisms use their sensory systems to acquire information from their environment and integrate this information to produce relevant behaviors. Nevertheless, how sensory information is converted into adequate motor patterns in the brain remains an open question. Here, we addressed this question using two-photon and light-sheet calcium imaging in intact, behaving zebrafish larvae. We monitored neural activity elicited by auditory stimuli while simultaneously recording tail movements. We observed a spatial organization of neural activity according to four different response profiles (frequency tuning curves), suggesting a low-dimensional representation of frequency information, maintained throughout the development of the larvae. Low frequencies (150-450 Hz) were locally processed in the hindbrain and elicited motor behaviors. In contrast, higher frequencies (900-1,000 Hz) rarely induced motor behaviors and were also represented in the midbrain. Finally, we found that the sensorimotor transformations in the zebrafish auditory system are a continuous and gradual process that involves the temporal integration of the sensory response in order to generate a motor behavior.

Bony labyrinth morphometry reveals hidden diversity in lungless salamanders (Family Plethodontidae): Structural correlates of ecology, development, and vision in the inner ear.

Lungless salamanders (Family Plethodontidae) form a highly speciose group that has undergone spectacular adaptive radiation to colonize a multitude of habitats. Substantial morphological variation in the otic region coupled with great ecological diversity within this clade make plethodontids an excellent model for exploring the ecomorphology of the amphibian ear. We examined the influence of habitat, development, and vision on inner ear morphology in 52 plethodontid species. We collected traditional and 3D geometric morphometric measurements to characterize variation in size and shape of the otic endocast and peripheral structures of the salamander ear. Phylogenetic comparative analyses demonstrate structural convergence in the inner ear across ecologically similar species. Species that dwell in spatially complex microhabitats exhibit robust, highly curved semicircular canals suggesting enhanced vestibular sense, whereas species with reduced visual systems demonstrate reduced canal curvature indicative of relaxed selection on the vestibulo-ocular reflex. Cave specialists show parallel enlargement of auditory-associated structures. The morphological correlates of ecology among diverse species reveal underlying evidence of habitat specialization in the inner ear and suggest that there exists physiological variation in the function of the salamander ear even in the apparent absence of selective pressures on the auditory system to support acoustic behavior.

Tetrapod-like pelvic girdle in a walking cavefish.

Fishes have adapted a number of different behaviors to move out of the water, but none have been described as being able to walk on land with a tetrapod-like gait. Here we show that the blind cavefish Cryptotora thamicola walks and climbs waterfalls with a salamander-like diagonal-couplets lateral sequence gait and has evolved a robust pelvic girdle that shares morphological features associated with terrestrial vertebrates. In all other fishes, the pelvic bones are suspended in a muscular sling or loosely attached to the pectoral girdle anteriorly. In contrast, the pelvic girdle of Cryptotora is a large, broad puboischiadic plate that is joined to the iliac process of a hypertrophied sacral rib; fusion of these bones in tetrapods creates an acetabulum. The vertebral column in the sacral area has large anterior and posterior zygapophyses, transverse processes, and broad neural spines, all of which are associated with terrestrial organisms. The diagonal-couplet lateral sequence gait was accomplished by rotation of the pectoral and pelvic girdles creating a standing wave of the axial body. These findings are significant because they represent the first example of behavioural and morphological adaptation in an extant fish that converges on the tetrapodal walking behaviour and morphology.

Hearing in Cavefishes.

Caves and associated subterranean habitats represent some of the harshest environments on Earth, yet many organisms, including fishes, have colonized and thrive in these habitats despite the complete absence of light, and other abiotic and biotic constraints. Over 170 species of fishes are considered obligate subterranean inhabitants (stygobionts) that exhibit some degree of troglomorphy, including degeneration of eyes and reduction in pigmentation. To compensate for lack of vision, many species have evolved constructive changes to non-visual sensory modalities. In this chapter we review hearing in cavefishes, with particular emphasize on our own studies on amblyopsid cavefishes. Hearing in cavefishes has not been well studied to date, as hearing ability has only been examined in four species. Two species show no differences in hearing ability relative to their surface relatives, while the other two species (family Amblyopsidae) exhibit regression in the form of reduced hearing range and reduction in hair cell densities on sensory epithelia. In addition to reviewing our current knowledge on cavefish hearing, we offer suggestions for future avenues of research on cavefish hearing and discuss the influence of Popper and Fay on the field of cavefish bioacoustics.

Genetically and environmentally mediated divergence in lateral line morphology in the Trinidadian guppy (Poecilia reticulata).

Fish and other aquatic vertebrates use their mechanosensory lateral line to detect objects and motion in their immediate environment. Differences in lateral line morphology have been extensively characterized among species; however, intraspecific variation remains largely unexplored. In addition, little is known about how environmental factors modify development of lateral line morphology. Predation is one environmental factor that can act both as a selective pressure causing genetic differences between populations, and as a cue during development to induce plastic changes. Here, we test whether variation in the risk of predation within and among populations of Trinidadian guppies (Poecilia reticulata) influences lateral line morphology. We compared neuromast arrangement in wild-caught guppies from distinct high- and low-predation population pairs to examine patterns associated with differences in predation pressure. To distinguish genetic and environmental influences, we compared neuromast arrangement in guppies from different source populations reared with and without exposure to predator chemical cues. We found that the distribution of neuromasts across the body varies between populations based on both genetic and environmental factors. To the best of our knowledge, this study is the first to demonstrate variation in lateral line morphology based on environmental exposure to an ecologically relevant stimulus.

Aerial jumping in the Trinidadian guppy (Poecilia reticulata).

Many fishes are able to jump out of the water and launch themselves into the air. Such behavior has been connected with prey capture, migration and predator avoidance. We found that jumping behavior of the guppy Poecilia reticulata is not associated with any of the above. The fish jump spontaneously, without being triggered by overt sensory cues, is not migratory and does not attempt to capture aerial food items. Here, we use high speed video imaging to analyze the kinematics of the jumping behavior P. reticulata. Fish jump from a still position by slowly backing up while using its pectoral fins, followed by strong body trusts which lead to launching into the air several body lengths. The liftoff phase of the jump is fast and fish will continue with whole body thrusts and tail beats, even when out of the water. This behavior occurs when fish are in a group or in isolation. Geography has had substantial effects on guppy evolution, with waterfalls reducing gene flow and constraining dispersal. We suggest that jumping has evolved in guppies as a behavioral phenotype for dispersal.

Evidence for hearing loss in amblyopsid cavefishes.

The constant darkness of caves and other subterranean habitats imposes sensory constraints that offer a unique opportunity to examine evolution of sensory modalities. Hearing in cavefishes has not been well explored, and here we show that cavefishes in the family Amblyopsidae are not only blind but have also lost a significant portion of their hearing range. Our results showed that cave and surface amblyopsids shared the same audiogram profile at low frequencies but only surface amblyopsids were able to hear frequencies higher than 800 Hz and up to 2 kHz. We measured ambient noise in aquatic cave and surface habitats and found high intensity peaks near 1 kHz for streams underground, suggesting no adaptive advantage in hearing in those frequencies. In addition, cave amblyopsids had lower hair cell densities compared with their surface relative. These traits may have evolved in response to the loud high-frequency background noise found in subterranean pools and streams. This study represents the first report of auditory regression in a subterranean organism.

Comparative genetics of the central nervous system in epigean and hypogean Astyanax mexicanus.

The extreme environment of subterranean caves presents an adaptive challenge to troglobitic organisms. The mechanisms by which natural selection modify an ancestral surface neural circuit to produce a novel subterranean behavior remain a mystery. To address this question, we performed cross species microarray experiments to compare differences in gene expression levels in the adult brain of the teleost Astyanax mexicanus. This species provides a unique opportunity for comparative genetic studies as it consists of extant epigean (surface) and hypogean (cave) conspecifics. Microarray experiments herein revealed significant changes in transcription levels of seventeen genes, several of which are important for behaviors involved in metabolic management. We focused on genes central to three neurotransmission and neuromodulation networks: the endocannabinoid system (Cannabinoid receptor CB1), the dopaminergic system (Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein) and the glutamatergic system (glutamate receptor AMPA 2a). All three genes were upregulated in the hypogean form of A. mexicanus compared to the epigean form, indicating that behavioral differences in the hypogean form of the species could be due to alterations in expression levels of several key genes. This information provides insights into the complex relationships among environmental factors, genetics, nervous systems and adaptive behavior, and can subsequently help us understand how these interactions affect behavior in other biological systems.

Evolution of a behavioral shift mediated by superficial neuromasts helps cavefish find food in darkness.

How cave animals adapt to life in darkness is a poorly understood aspect of evolutionary biology [1]. Here we identify a behavioral shift and its morphological basis in Astyanax mexicanus, a teleost with a sighted surface-dwelling form (surface fish) and various blind cave-dwelling forms (cavefish) [2-4]. Vibration attraction behavior (VAB) is the ability of fish to swim toward the source of a water disturbance in darkness. VAB was typically seen in cavefish, rarely in surface fish, and was advantageous for feeding success in the dark. The potential for showing VAB has a genetic component and is linked to the mechanosensory function of the lateral line. VAB was evoked by vibration stimuli peaking at 35 Hz, blocked by lateral line inhibitors, first detected after developmental increases in superficial neuromast (SN) number and size [5-7], and significantly reduced by bilateral ablation of SN. We conclude that VAB and SN enhancement coevolved to compensate for loss of vision and to help blind cavefish find food in darkness.

Detection of interaural time differences in the alligator.

The auditory systems of birds and mammals use timing information from each ear to detect interaural time difference (ITD). To determine whether the Jeffress-type algorithms that underlie sensitivity to ITD in birds are an evolutionarily stable strategy, we recorded from the auditory nuclei of crocodilians, who are the sister group to the birds. In alligators, precisely timed spikes in the first-order nucleus magnocellularis (NM) encode the timing of sounds, and NM neurons project to neurons in the nucleus laminaris (NL) that detect interaural time differences. In vivo recordings from NL neurons show that the arrival time of phase-locked spikes differs between the ipsilateral and contralateral inputs. When this disparity is nullified by their best ITD, the neurons respond maximally. Thus NL neurons act as coincidence detectors. A biologically detailed model of NL with alligator parameters discriminated ITDs up to 1 kHz. The range of best ITDs represented in NL was much larger than in birds, however, and extended from 0 to 1000 micros contralateral, with a median ITD of 450 micros. Thus, crocodilians and birds employ similar algorithms for ITD detection, although crocodilians have larger heads.

Interaural timing difference circuits in the auditory brainstem of the emu (Dromaius novaehollandiae).

In the auditory system, precise encoding of temporal information is critical for sound localization, a task with direct behavioral relevance. Interaural timing differences (ITDs) are computed using axonal delay lines and cellular coincidence detectors in nucleus laminaris (NL). We present morphological and physiological data on the timing circuits in the emu, Dromaius novaehollandiae, and compare these results with those from the barn owl (Tyto alba) and the domestic chick (Gallus gallus). Emu NL was composed of a compact monolayer of bitufted neurons whose two thick primary dendrites were oriented dorsoventrally. They showed a gradient in dendritic length along the presumed tonotopic axis. The NL and nucleus magnocellularis (NM) neurons were strongly immunoreactive for parvalbumin, a calcium-binding protein. Antibodies against synaptic vesicle protein 2 and glutamic acid decarboxlyase revealed that excitatory synapses terminated heavily on the dendritic tufts, while inhibitory terminals were distributed more uniformly. Physiological recordings from brainstem slices demonstrated contralateral delay lines from NM to NL. During whole-cell patch-clamp recordings, NM and NL neurons fired single spikes and were doubly rectifying. NL and NM neurons had input resistances of 30.0 +/- 19.9 Momega and 49.0 +/- 25.6 Momega, respectively, and membrane time constants of 12.8 +/- 3.8 ms and 3.9 +/- 0.2 ms. These results provide further support for the Jeffress model for sound localization in birds. The emu timing circuits showed the ancestral (plesiomorphic) pattern in their anatomy and physiology, while differences in dendritic structure compared to chick and owl may indicate specialization for encoding ITDs at low best frequencies.

The lens has a specific influence on optic nerve and tectum development in the blind cavefish Astyanax.

We used the teleost Astyanaxmexicanus to examine the role of the lens in optic nerve and tectum development. This speciesis unusually suited for studies of nervous system development and evolution because of its two extant forms: an eyed surface dwelling (surface fish) and several blind cave dwelling (cavefish) forms. Cavefish embryos initially form eye primordia, but the lens eventually dies by apoptosis, then the retina ceases to grow, and finally the degenerating eyes sink into the orbits. Transplantation of an embryonic surface fish lens into a cavefish optic cup restores eye development. We show here that retinal nerve fibers are formed and project to the optic tectum in cavefish embryos. In adult cavefish that have completed lens degeneration, however, the number of retinal axons in the optic nerve is substantially reduced compared to surface fish. The presumptive brain domains of embryonic cavefish are not altered relative to surface fish based on expression of the regional marker genes Pax6, Pax2.1, and engrailed2. In contrast, the adult cavefish brain is elongated, the optic tectum is diminished in volume, and the number of tectal neurons is reduced relative to surface fish. Unilateral transplantation of an embryonic surface fish lens into a cavefish optic cup increases the size of the optic nerve, the number of retinotectal projections from the restored eye, and the volume and neuronal content of the contralateral optic tectum. The results suggest that the lens has a specific influence on optic nerve and tectum development during eye growth in Astyanax.

Intrinsic neuronal properties of the chick nucleus angularis.

In vitro whole cell recording revealed intrinsic firing properties and single-cell morphology in the cochlear nucleus angularis (NA) of the chick. We classified three major classes of neurons: one-spike, damped, and tonic. A delayed inward rectifying current was observed in all classes during hyperpolarization injections. One-spike neurons responded with a single spike to depolarizing current injection and had small (stubby) radiate dendritic trees. Damped neurons responded with only a few spikes at the onset of positive current injection. More positive current inputs led to a damped response. Damped cell dendrites had a planar orientation parallel to the isofrequency axis in NA. Tonic cells produced trains of action potentials in response to a depolarizing current injection. Three variations of the tonic type had multipolar morphology, with dendrites oriented either radially (I and III) or perpendicular to the tonotopic axis (II; vertical). Tonics I and III differed in the shape of their action potential undershoot. Thus NA is both physiologically and morphologically heterogeneous.