Secondary Sensory Cells Research Articles

Sensory cells in tunicates: insights into mechanoreceptor evolution.

Tunicates, the sister group of vertebrates, offer a unique perspective for evolutionary developmental studies (Evo-Devo) due to their simple anatomical organization. Moreover, the separation of tunicates from vertebrates predated the vertebrate-specific genome duplications. As adults, they include both sessile and pelagic species, with very limited mobility requirements related mainly to water filtration. In sessile species, larvae exhibit simple swimming behaviors that are required for the selection of a suitable substrate on which to metamorphose. Despite their apparent simplicity, tunicates display a variety of mechanoreceptor structures involving both primary and secondary sensory cells (i.e., coronal sensory cells). This review encapsulates two decades of research on tunicate mechanoreception focusing on the coronal organ's sensory cells as prime candidates for understanding the evolution of vertebrate hair cells of the inner ear and the lateral line organ. The review spans anatomical, cellular and molecular levels emphasizing both similarity and differences between tunicate and vertebrate mechanoreception strategies. The evolutionary significance of mechanoreception is discussed within the broader context of Evo-Devo studies, shedding light on the intricate pathways that have shaped the sensory system in chordates.

Sensory Hair Cells: An Introduction to Structure and Physiology.

Sensory hair cells are specialized secondary sensory cells that mediate our senses of hearing, balance, linear acceleration, and angular acceleration (head rotation). In addition, hair cells in fish and amphibians mediate sensitivity to water movement through the lateral line system, and closely related electroreceptive cells mediate sensitivity to low-voltage electric fields in the aquatic environment of many fish species and several species of amphibian. Sensory hair cells share many structural and functional features across all vertebrate groups, while at the same time they are specialized for employment in a wide variety of sensory tasks. The complexity of hair cell structure is large, and the diversity of hair cell applications in sensory systems exceeds that seen for most, if not all, sensory cell types. The intent of this review is to summarize the more significant structural features and some of the more interesting and important physiological mechanisms that have been elucidated thus far. Outside vertebrates, hair cells are only known to exist in the coronal organ of tunicates. Electrical resonance, electromotility, and their exquisite mechanical sensitivity all contribute to the attractiveness of hair cells as a research subject.

Distribution of olfactory marker protein in the rat vomeronasal organ

Olfactory marker protein (OMP) may act as a modulator within the olfactory signal-transduction cascade. It has also been shown to have some importance in development of olfactory sensory organs. Here we used high resolution immunocytochemistry to localize OMP in the rat vomeronasal organ (VNO). Immunofluorescence for OMP was abundant in cilia and in apical dendrites of sensory cells, mostly associated with intraepithelial capillaries. Perikarya were stained to a lesser extent while intense OMP immunoreactivity was seen in axons of sensory neurons. Single cells within the non-sensory portion of the VNO exhibited intense OMP immunofluorescence in apical cilia and weak cytoplasmic staining. Some of the exocrine cells in the vomeronasal glands contained OMP positive secretory granules. Electron microscopy revealed that non-sensory ciliated cells had short rod like kinocilia as well as microvilli. These cells contained secretory vesicles. Their basal portion was in close apposition to nerve endings. Our findings suggest that the sensory part of the VNO contains OMP positive sensory neurons and that the non-sensory epithelium may contain secondary sensory cells. In addition OMP may be liberated from secretory glands into vomeronasal secretions.

Cytodifferentiation of hair cells during the development of a basal chordate

Tunicates are unique animals for studying the origin and evolution of vertebrates because they are considered vertebrates' closest living relatives and share the vertebrate body plan and many specific features. Both possess neural placodes, transient thickenings of the cranial ectoderm that give rise to various types of sensory cells, including axonless secondary mechanoreceptors. In vertebrates, these are represented by the hair cells of the inner ear and the lateral line, which have an apical apparatus typically bearing cilia and stereovilli. In tunicates, they are found in the coronal organ, which is a mechanoreceptor located at the base of the oral siphon along the border of the velum and tentacles and is formed of cells bearing a row of cilia and short microvilli. The coronal organ represents the best candidate homolog for the vertebrate lateral line. To further understand the evolution of secondary sensory cells, we analysed the development and cytodifferentiation of coronal cells in the tunicate ascidian Ciona intestinalis for the first time. Here, coronal sensory cells can be identified as early as larval metamorphosis, before tentacles form, as cells with short cilia and microvilli. Sensory cells gradually differentiate, acquiring hair cell features with microvilli containing actin and myosin VIIa; in the meantime, the associated supporting cells develop. The coronal organ grows throughout the animal's lifespan, accompanying the growth of the tentacle crown. Anti-phospho Histone H3 immunostaining indicates that both hair cells and supporting cells can proliferate. This finding contributes to the understanding of the evolution of secondary sensory cells, suggesting that both ancestral cell types were able to proliferate and that this property was progressively restricted to supporting cells in vertebrates and definitively lost in mammals.

Evolutionary diversification of secondary mechanoreceptor cells in tunicata.

BackgroundHair cells are vertebrate secondary sensory cells located in the ear and in the lateral line organ. Until recently, these cells were considered to be mechanoreceptors exclusively found in vertebrates that evolved within this group. Evidence of secondary mechanoreceptors in some tunicates, the proposed sister group of vertebrates, has recently led to the hypothesis that vertebrate and tunicate secondary sensory cells share a common origin. Secondary sensory cells were described in detail in two tunicate groups, ascidians and thaliaceans, in which they constitute an oral sensory structure called the coronal organ. Among thaliaceans, the organ is absent in salps and it has been hypothesised that this condition is due to a different feeding system adopted by this group of animals. No information is available as to whether a comparable structure exists in the third group of tunicates, the appendicularians, although different sensory structures are known to be present in these animals.ResultsWe studied the detailed morphology of appendicularian oral mechanoreceptors. Using light and electron microscopy we could demonstrate that the mechanosensory organ called the circumoral ring is composed of secondary sensory cells. We described the ultrastructure of the circumoral organ in two appendicularian species, Oikopleura dioica and Oikopleura albicans, and thus taxonomically completed the data collection of tunicate secondary sensory cells. To understand the evolution of secondary sensory cells in tunicates, we performed a cladistic analysis using morphological data. We constructed a matrix consisting of 19 characters derived from detailed ultrastructural studies in 16 tunicate species and used a cephalochordate and three vertebrate species as outgroups.ConclusionsOur study clearly shows that the circumoral ring is the appendicularian homologue of the coronal organ of other tunicate taxa. The cladistic analysis enabled us to reconstruct the features of the putative ancestral hair cell in tunicates, represented by a simple monociliated cell. This cell successively differentiated into the current variety of oral mechanoreceptors in the various tunicate lineages. Finally, we demonstrated that the inferred evolutionary changes coincide with major transitions in the feeding strategies in each respective lineage.

Variability of hair cells in the coronal organ of ascidians (Chordata, Tunicata)

The tunicate ascidians are nonvertebrate chordates that possess mechanoreceptor cells in the coronal organ in the oral siphon, which monitor the incoming water flow. Like vertebrate hair cells, the mechanoreceptor–coronal cells are secondary sensory (axonless) cells accompanied by supporting cells and they exhibit morphological diversities of apical specialisations: they are multiciliate in ascidians of the order Enterogona, whereas they are more complex and possess one or two cilia accompanied by stereovilli, also graded in length, in ascidians of the order Pleurogona. In morphology, embryonic origin, and arrangement, coronal sensory cells closely resemble vertebrate hair cells. We describe here the coronal organs of five ascidians ( Pyura haustor (Stimpson, 1864), Pyura stolonifera (Heller, 1878), Styela gibbsii (Stimpson, 1864), Styela montereyensis (Dall, 1872), and Polyandrocarpa zorritensis (Van Name, 1931)), belonging to Pleurogona, also comprising species of one family (Pyuridae), not yet considered, and thus completing our overview of the order. Each species possesses at least two kinds of secondary sensory cells, some of them characterized by stereovilli graded in length. In some species, the coronal sensory cells exhibit secretory activity; in P. haustor, a mitotic sensory cell has also been found. We compare the coronal organ in both ascidians and with other chordate sensory organs formed of secondary sensory cells, and discuss their possible homologies.

Hair cells in non-vertebrate models: Lower chordates and molluscs

The study of hair cells in invertebrates is important, because it can shed light on the debated question about the evolutionary origin of vertebrate hair cells. Here, we review the morphology and significance of hair cells in two groups of invertebrates, the lower chordates (tunicates and cephalochordates) and the molluscs. These taxa possess complex mechanoreceptor organs based on both primary (sensory neurons) and/or secondary, axonless, sensory cells, bearing various apical specializations. Compared with vertebrates, these taxa show interesting examples of convergent evolution and possible homologies of sensory systems. For example, the “lateral line organ” of Octopoda and Decapoda, composed of primary sensory cells aligned on the arms and the head, is considered a classic example of convergent evolution to mechanoreception. Similarly, in ascidians, the cupular organ, formed of primary sensory cells embedded in a gelatinous cupula, is seen as an analog of neuromasts in vertebrates. However, the coronal organ of the oral siphon of ascidians, represented by a line of secondary sensory cells with a hair bundle also comprising graded stereovilli, is currently the best candidate for tracing the evolutionary origin of the vertebrate octavo-lateralis system. Several features, such as embryological origin, position, gene expression and morphology, support this hypothesis.

Does hair cell differentiation predate the vertebrate appearance?

It is generally accepted that the three main chordate groups (tunicates, cephalochordates and vertebrates) originated from a common ancestor having the basic features of the chordate body plan, i.e. a neural tube and a notochord flanked by striated musculature. There is now increasing evidence that tunicates, rather than cephalochordates, are the vertebrate sister-group. Correlated with this, tunicates have sensory structures similar to those derived from placodes or neural crest in vertebrates. In this context, we discuss here whether the precursors of vertebrate hair cells, which are placodal in origin, were present in ancestral chordates. The ascidian tunicates possess a coronal organ, consisting of a row of mechanosensory cells that runs around the base of the oral siphon. Its function is to monitor the incoming water flow. The cells are secondary sensory cells, i.e. they lack axons and synapse with neurons whose somata lie in the cerebral ganglion. They are accompanied by supporting cells and, as in vertebrates, have varying morphologies in the species so far examined: in one order (Enterogona), they are multiciliate; in the other (Pleurogona), they may possess an apical apparatus, consisting of one or two cilia accompanied by stereovilli, that are graded in length. Coronal cells thus resemble vertebrate hair cells closely in their morphology, embryonic origin and arrangement, which suggests they originated early in ancestral chordates. We are continuing our study of the coronal organ in other ascidian species, and report new data here on Botrylloides leachi, which conforms with the pattern of Pleurogona and, in particular, with previously published results on other botryllid ascidians.

Hair cells in an ascidian (Tunicata) and their evolution in chordates

In ascidians, mechanoreceptors of the oral area are involved in monitoring the incoming water flow. Sensory cells are represented by scattered, ciliated primary cells (sending their own axons to the cerebral ganglion) or secondary sensory cells (axonless cells forming afferent and efferent synapses with neurons, whose somata are located in the ganglion) of the coronal organ. Coronal cells have varying morphologies: in species of the Enterogona order, they are multiciliate, whereas those of Pleurogona possess an apical apparatus composed of one or two cilia accompanied by stereovilli, in some cases also graded in length. The coronal organ has been proposed as a homologue to the vertebrate octavo-lateralis system, because coronal cells resemble vertebrate hair cells for morphology, embryonic origin and arrangement. In the ascidian Molgula socialis (Pleurogona), we now describe the morphology of the coronal organ, which contains a few associated rows of sensory cells that run the whole length of the oral velum and the branched tentacles. Three kinds of sensory cells, accompanied by specialised supporting cells, are present. Comparisons between the coronal organ and other chordate mechanosensory structures suggest that hair cells originated in the common ancestor of chordates.

Coronal organ of ascidians and the evolutionary significance of secondary sensory cells in chordates

A new mechanoreceptor organ, the coronal organ, in the oral siphon of some ascidians belonging to the order Pleurogona has recently been described. In contrast to the known mechanoreceptor organs of ascidian atrium that consist of sensory neurons sending their own axons to the cerebral ganglion, coronal sensory cells are secondary mechanoreceptors, i.e., axonless cells forming afferent and efferent synapses with neurites of neurons located in the ganglion. Moreover, coronal cells exhibit an apical apparatus composed of a cilium accompanied or flanked by rod-like microvilli (stereovilli). Because of the resemblance of these cells to vertebrate hair cells, their ectodermal origin and location in a linear array bordering the bases of the oral tentacles and velum, the coronal organ has been proposed as a homologue to the vertebrate acousticolateralis system. Here we describe the morphology of the coronal organs of six ascidians belonging to the suborders Phlebobranchia and Aplousobranchia (order Enterogona). The sensory cells are ciliated, lack typical stereovilli, and at their bases form synapses with neurites. In two species, the sensory cells are accompanied by large cells involved in synthesis and secretion of protein. We hypothesize that the coronal organ with its secondary sensory cells represents a plesiomorphic feature of ascidians. We compare the coronal organ with other chordate sensory organs formed of secondary sensory cells, i.e., the ventral lip receptors of appendicularians, the oral secondary sensory cells of cephalochordates, and the acousticolateralis system of vertebrates, and we discuss their homologies at different levels of organization.

Novel, secondary sensory cell organ in ascidians: in search of the ancestor of the vertebrate lateral line.

A new mechanoreceptor organ, the "coronal organ," located in the oral siphon, is described by light and electron microscopy in the colonial ascidians Botryllus schlosseri and Botrylloides violaceus. It is composed of a line of sensory cells (hair cells), accompanied by supporting cells, that runs continuously along the margin of the velum and tentacles of the siphon. These hair cells resemble those of the vertebrate lateral line or, in general, the acoustico-lateralis system, because they bear a single cilium, located centrally or eccentrically to a hair bundle of numerous stereovilli. In contrast to other sensory cells of ascidians, the coronal hair cells are secondary sensory cells, since they lack axonal processes directed towards the cerebral ganglion. Moreover, at their base they form synapses with nerve fibers, most of which exhibit acetylcholinesterase activity. The absence of axonal extensions was confirmed by experiments with lipophilic dyes. Different kinds of synapses were recognized: usually, each hair cell forms a few afferent synapses with dendrites of neurons located in the ganglion; efferent synapses, both axo-somatic (between an axon coming from the ganglion and the hair cell) and axo-dendritic (between an axon coming from the ganglion and an afferent fiber) were occasionally found. The presence of secondary sensory cells in ascidians is discussed in relation to the evolution of sensory cells and placodes in vertebrates. It is proposed that the coronal organ in urochordates is homologous to the vertebrate acoustico-lateralis system.

Neurotrophin 3 potentiates glutamatergic responses of IHC afferents in the cochlea in vivo.

Neurotrophins have traditionally been regarded as slow-acting signals essential for neuronal survival and differentiation. Recent studies with neuronal slices, cultures and nerve ending preparations have shown that neurotrophins generate acute changes in nerve activity. Among the secondary sensory cells are the inner hair cells (IHC) and taste buds, cells which express the neurotrophic factors necessary for the survival of their innervating neurons. If in these cells neurotrophins acutely affect the nerve activity of their afferent neurons, as in the central nervous system (CNS), this may have important functional implications for the corresponding sensory transduction processes. The neurotrophin NT-3 has been reported to be expressed in IHCs. We chose an in vivo application system for the microiontophoretic supply of NT-3 in the subsynaptic region of the IHC. The effect of NT-3 on spontaneous and evoked afferent cochlear nerve activities in adult guinea pig inner ear was studied. We observed that NT-3 rapidly increases the spontaneous and glutamate-evoked firing rate of IHC afferents. Moreover, firing induced by both N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) were specifically enhanced during the presence of NT-3, a process which was selectively blocked by the tyrosine kinase receptor inhibitor K252a. Because we localized NT-3 mRNA not only in IHCs but also in the spiral ganglion, we propose that similar to other sensory systems, afferent and autocrine neurotrophin activities may be responsible for survival of cochlear neurons. In addition, NT-3 in IHCs may operate as a signal-dependent, intrinsic neuromodulator and/or neuroprotector.

A reexamination of the epithelial sensory cells of amphioxus (Branchiostoma)

The rostral epithelium of a newly metamorphosed juvenile of Branchiostoma floridae was examined at the EM level to confirm previous reports on its sensory cells. The majority of the sensory cells are of three types: two type I variants, with simple collars of unbranched microvilli surrounding their cilia, and one kind of type II cell, with an extended collar of repeatedly branched microvilli. The two type I variants differ in the structure and arrangement of the microvilli, basal body and rootlet, and the length of the cilium. Both variants are probably primary sensory cells (i.e. each has its own axon), but the data supporting this conclusion are much better for one variant than for the other. Type II cells are secondary sensory cells, with synaptic terminals borne on short extensions of the cell body. The presence of degenerating type II cells suggests that they may be subject to a regular process of loss and renewal. The results do not resolve the evolutionary issue of how amphioxus sensory cells relate to the epithelial sensory and receptor cells of vertebrates. Being primary, the type I cells resemble the supposed ancestral type more closely than do type II cells. Type II cells may be chemosensory, however, and should not be ruled out a priori as possible homologues of either primary or secondary chemosensory cells in vertebrates.

High-Voltage Electron Microscopy and 3-D Reconstruction of Solitary Chemosensory Cells in the Anterior Dorsal Fin of the Gadid FishCiliata mustela(Teleostei)

Solitary chemosensory cells (SCCs) are secondary sensory cells present in the epidermis of most primary aquatic vertebrates. In rocklings, the epidermis of the anterior dorsal fin (ADF) contains approximately 5 million SCCs. High-voltage electron microscopy and three-dimensional reconstructions from serial sections were used to examine the ultrastructure, arrangement, and synaptic contacts of the SCCs in the rockling ADF. Approximately 15% of all cells in the fin ray epidermis are SCCs, which occupy roughly 30% of the epidermal volume. These spindle-shaped cells are 25–30 μm long and up to 10 μm wide and terminate apically in a microvillus protruding 2–5 μm above the epidermal surface. SCCs contain abundant endoplasmic reticulum and a large Golgi apparatus in their proximal regions. The distal parts of SCCs contain characteristic vesicles, elongate mitochondria, and longitudinal strands of intermediate filaments. Synapses between SCCs and nerves resemble those found in teleost taste buds. One to four synaptic contacts per SCC were found. We hypothesize that the apparent secretory activity of the SCCs serves to replenish the apical membrane and mucus. Furthermore, parallel sampling of several hundred SCCs by single nerve fibers may serve low-threshold detection rather than stimulus localization.

Synaptic interactions between crista hair cells in the statocyst of the squid Alloteuthis subulata.

Intracellular injections of the fluorescent dye Lucifer yellow into the various cell types within the anterior transverse crista segment of the statocyst of squid revealed that the primary sensory hair cells and both large and small first-order afferent neurons have relatively simple morphologies, each cell having a single, unbranched axon that passes directly into the small crista nerve that innervates the anterior transverse crista. However, the small first-order neurons have short dendritic processes occurring in the region of the sensory hair cells. The secondary sensory hair cells have no centripetal axons, but some have long processes extending from their bases along the segment. Simultaneous intracellular recordings from pairs of the different cell types in the anterior transverse crista segment demonstrated that electrical coupling is widespread; secondary sensory hair cells are coupled electrically along a hair cell row, as are groups of primary sensory hair cells. Secondary sensory hair cell also are coupled to neighboring small first-order afferent neurons. However, this coupling is rectifying in that it only occurs from secondary sensory hair cells to first-order afferent neurons. Direct electrical stimulation of the small crista nerve to excite the efferent axons revealed efferent connections to both the primary sensory hair cells and the small first-order afferent neurons. These efferent responses were of three types: excitatory or inhibitory postsynaptic potentials and excitatory postsynaptic potentials followed by inhibitory postsynaptic potentials. The functional significance of the cell interactions within the crista epithelium of the statocyst of squid is discussed and comparisons drawn with the balance organs of other animals.

The Merkel cell as a possible mechanoreceptor cell

Merkel cells (MCs) are abundant at the basal layer of various skin in vertebrates, and make synaptic contacts with nerve endings to form the Merkel cell-neurite complex (MCN-complex). It has been established that the MCN-complex is involved in slowly adapting mechanoreception, cutaneous afferents of which are called SAI units in mammals or Ft-I units in frogs. However, the MC function has been the focus of attention, and some hypotheses propose that the site of mechanoreception is at the nerve terminals but not at the MC. In the present review, the possibility that MCs are the mechanoreceptors was focused on recent findings. Irradiation of quinacrine-loaded MCs in the rat hairy skin using excitation light degenerates the MCs selectively with the nerve terminals left intact. Correspondingly, SAI units decrease tonic discharges rapidly, but phasic responses remain intact. Blocking synaptic transmission in the MCN-complexes by divalent or alkyl Ca antagonists in mammals or frogs heavily decreases the tonic mechanical responses of the afferent units, but the phasic responses are rather resistant. Application of anodal current on the Ft-I receptive spots produces tonic discharges as in hair cells or taste cells, while the threshold to elicit the first spike is lower with cathodal than anodal stimulation, in contrast with other secondary sensory cells. These findings indicate that MCs are mechanoreceptors to yield tonic responses, while the nerve terminals may transduce the transient phase. Further studies, particularly on mechanically-gated ionic channels in the MC membrane and on transmitters between the MCs and nerve terminal, are necessary to establish the MC as mechanoreceptors.

Ionic currents in secondary sensory hair cells isolated from the statocysts of squid and cuttlefish

Mechanosensitive hair cells in the statocysts of cephalopods underlie a sophisticated detection system for linear and angular accelerations. To investigate the operation of this system, secondary sensory hair cells were dissociated from the sensory epithelia of these statocysts and their voltage sensitive ionic conductances identified and characterized under whole cell voltage clamp.

The responses of primary and secondary sensory hair cells in the squid statocyst to mechanical stimulation

Intracellular recordings were obtained from primary and secondary sensory hair cells in the anterior transverse crista segment of the squid (Alloteuthis subulata) statocyst during imposed displacements of the overlying cupula. The secondary sensory hair cells were depolarized by ventral movements of the cupula and hyperpolarized by dorsal cupula movements. The displacement/response curve was asymmetric around the zero position and sigmoidal in shape, similar to that already described for vertebrate hair cells. The cells are estimated to have a sensitivity of at least 0.5 mV per degree angle of cilia displacement. The responses showed pronounced adaptation and could be blocked by bath applied alcohols, such as heptanol or octanol, or by high concentrations of aminoglycosides.

A Simple, Reliable and Inexpensive Silver Stain for Nerve Fibers in Bleached Skin

A procedure for silver staining is described which leads to the selective and reliable impregnation of nerve fibers in bleached skin of vertebrates and invertebrates. In combination with osmium, the protocol enhances the staining of secondary sensory cells of mechanosensory and electrosensory organs so that the innervation pattern of each organ and the number of sensory cells per organ can easily be evaluated. The technique can be also used for staining nerve fibers in whole embryos.

The angular acceleration receptor system of the statocyst of Octopus vulgaris : morphometry, ultrastructure, and neuronal and synaptic organization

The angular acceleration receptor system (crista/cupula system) of the statocyst of Octopus vulgaris has been thoroughly reinvestigated, and detailed information is presented regarding its morphometry, ultrastructure, and neuronal and synaptic organization. In each of the nine crista sections, some receptor hair cells are primary sensory cells with an axon extending from their base. Also, there are large and small secondary sensory hair cells without axons, which make afferent synapses with large and small first-order afferent neurons. The afferent synapses are of two morphologically distinct types, having either a finger-like or a flat postsynaptic process; both can be seen in the same hair cell. In addition to the afferents, there is a dense plexus of efferent fibres in each crista section, and efferent synapses can be seen at the level of the hair cells and of the neurons. The morphometric analysis of the nine crista sections shows obvious differences between the odd-numbered (C1, C3, C5, C7, C9) and the even-numbered (C2, C4, C6, C8) crista sections: they differ in length, in the number of the small primary sensory cells and in the number of the small first-order afferent neurons. Centrifugal cobalt filling of the three crista nerves revealed a disproportionate innervation of the nine crista sections: the anterior crista nerve innervates section C1 and the first half of section C2, the medial crista nerve innervates the second half of section C2, sections C3, C4, C5, and the first half of section C6, and the posterior crista nerve innervates the second half of section C6, and sections C7, C8 and C9. In each of the three crista nerves, only 25% of the total number of axons are afferent fibres, the remaining 75 % are efferent. To each of the nine crista sections a cupula is attached. In the form and size of the cupulae there is again a conspicuous difference between the odd and the even crista sections: a small widebased cupula is attached to each of the odd crista sections, whereas the even crista sections each have a large narrow-based cupula with a small area of attachment. The results are discussed with reference to their functional consequences.