Adaptations of the vestibular system to short and long-term exposures to altered gravity.

Long-term space flight creates unique environmental conditions to which the vestibular system must adapt for optimal survival of a given organism. The development and maintenance of vestibular connections are controlled by environmental gravitational stimulation as well as genetically controlled molecular interactions. This paper describes the effects of hypergravity on axonal growth and dendritic morphology, respectively. Two aspects of this vestibular adaptation are examined: (1) How does long-term exposure to hypergravity affect the development of vestibular axons? (2) How does short-term exposure to extremely rapid changes in gravity, such as those that occur during shuttle launch and landing, affect dendrites of the vestibulocerebellar system? To study the effects of longterm exposures to altered gravity, embryonic rats that developed in hypergravity were compared to microgravity-exposed and control rats. Examination of the vestibular projections from epithelia devoted to linear and angular acceleration revealed that the terminal fields segregate differently in rat embryos that gestated in each of the gravitational environments.To study the effects of short-term exposures to altered gravity, mice were exposed briefly to strong vestibular stimuli and the vestibulocerebellum was examined for any resulting morphological changes. My data show that these stimuli cause intense vestibular excitation of cerebellar Purkinje cells, which induce up-regulation of clathrin-mediated endocytosis and other morphological changes that are comparable to those seen in long-term depression. This system provides a basis for studying how the vestibular environment can modify cerebellar function, allowing animals to adapt to new environments.

Development of the ear and of connections between the ear and the brain: is there a role for gravity?

This paper outlines the development of the gravistatic sensory system of the ear. First, evidence is presented that a genetic program, for which major transcription factors have already been identified using gene expression studies and targeted mutagenesis, governs the initial development of this system. Second, the formation of sensory neurons and their connections to the brain is described as revealed by tracing studies and genetic manipulations. It is concluded that the initial development of the connections of sensory neurons with mechanosensory transducers of the ear (the hair cells) and the targets in the brainstem (vestibular nuclei) is also dependent on fairly rigid genetic programs. During late embryonic and early postnatal development, however, sensory input appears to be used to fine-tune connections of these sensory neurons with the hair cells in the ear as well as with second order vestibular neurons in the brainstem. This phase is proposed to be critical for a proper calibration of the gravistatic information processing in the brain.

Postnatal development of efferent synapses in the rat cochlea.

The development of olivocochlear efferent axons and their contacts in the postnatal cochlea was studied after DiI applications to the olivocochlear bundle in the ipsilateral brainstem of rats from 0 to 10 days of age (P0-10). Light microscopic analyses showed that labeled axons reached the vicinity of inner hair cells by P0 and outer hair cells by P2. Electron microscopic analyses demonstrated that labeled immature efferent axons are present among supporting cells of the greater epithelial ridge as well as inner hair cells at P0. The first efferent contacts that contacted inner hair cells contained a few irregularly sized vesicles and, occasionally, mitochondria. Postsynaptic specializations within inner hair cells apposed to labeled efferent axons included subsynaptic cisterns, irregularly sized vesicles, and synaptic bodies. Similar features were present in unlabeled profiles, presumed to be afferents, indicating that immature efferent axons could not be reliably distinguished from afferents without positive labeling. Efferent axons synapsed with outer hair cells by P4 and had synapse-like contacts at the bases of Deiters' cells at P4 and P6. Contacts between afferents and efferents were observed frequently in the inner spiral bundle from P6. As they matured, efferent axon terminals contacting hair cells contained increasing numbers of synaptic vesicles and were typically apposed by well-defined postsynaptic cisterns, thus acquiring distinctive profiles.

Effects of microgravity on vestibular development and function in rats: genetics and environment.

Our anatomical and behavioral studies of embryonic rats that developed in microgravity suggest that the vestibular sensory system, like the visual system, has genetically mediated processes of development that establish crude connections between the periphery and the brain. Environmental stimuli also regulate connection formation including terminal branch formation and fine-tuning of synaptic contacts. Axons of vestibular sensory neurons from gravistatic as well as linear acceleration receptors reach their targets in both microgravity and normal gravity, suggesting that this is a genetically regulated component of development. However, microgravity exposure delays the development of terminal branches and synapses in gravistatic but not linear acceleration-sensitive neurons and also produces behavioral changes. These latter changes reflect environmentally controlled processes of development.

The development of corticocollicular projections in anophthalmic mice.

To determine the role of retinal axons in the development of the corticocollicular projection in mice, the lipophilic fluorescent dye, DiI, was used to compare the development of the cortical projections in phenotypically normal (C57BL/6J) mice to that of anophthalmic 129SV/CPorJ mice. Cortical axons in anophthalmic mice found their targets and established a laminar specificity similar to those of cortical axons in normal mice despite the absence of the retinal projection. Cortical axons in normal mice reached the superior colliculus before those in anophthalmic mice and also had a faster rate of growth within the colliculus. Unlike cortical axons in normal mice in early postnatal ages, those in anophthalmic mice formed a disperse bundle in the stratum opticum. Axons labeled by focal applications of DiI into area 17 terminated in a larger and more medial area in anophthalmic mice than in normal mice. Thus, retinal axons are not essential for cortical axons to reach the superior colliculus, but they may have a role in organizing the growth of later-arriving cortical axons. Furthermore, cortical axons can terminate in the superior colliculus with a coarse topography when retinal axons are absent, but they cannot form a topographically refined projection.

The maturation of corticocollicular neurons in mice.

The location and development of the neurons that give rise to the corticocollicular projection were studied in C57BL/6J and 129SV/CPorJ anophthalmic mice. The first neurons that project to the superior colliculus appear in the subplate zone at E13 in C57BL/6J mice. Cortical plate neurons reach the colliculus about 2 days later. The appearance and development of these neurons are delayed by about 2 days in the anophthalmic strain.

The development of vestibulocochlear efferents and cochlear afferents in mice.

We have reinvestigated the embryonic development of the vestibulocochlear system in mice using anterograde and retrograde tracing techniques. Our studies reveal that rhombomeres 4 and 5 include five motor neuron populations. One of these, the abducens nucleus, will not be dealt with here. Rhombomere 4 gives rise to three of the remaining populations: the facial branchial motor neurons; the vestibular efferents; and the cochlear efferents. The migration of the facial branchial motor neurons away from the otic efferents is completed by 13.5 days post coitum (dpc). Subsequently the otic efferents separate into the vestibular and cochlear efferents, and complete their migration by 14.5 dpc. In addition to their common origin, all three populations have perikarya that migrate via translocation through secondary processes, form a continuous column upon completion of their migrations, and form axonal tracts that run in the internal facial genu. Some otic efferent axons travel with the facial branchial motor nerve from the internal facial genu and exit the brain with that nerve. These data suggest that facial branchial motor neurons and otic efferents are derived from a common precursor population and use similar cues for pathway recognition within the brain. In contrast, rhombomere 5 gives rise to the fourth population to be considered here, the superior salivatory nucleus, a visceral motor neuron group. Other differences between this group and those derived from rhombomere 4 include perikaryal migration as a result of translocation first through primary processes and only then through secondary processes, a final location lateral to the branchial motor/otic efferent column, and axonal tracts that are completely segregated from those of the facial branchial and otic efferents throughout their course inside the brain. Analysis of the peripheral distribution of the cochlear efferents and afferents show that efferents reach the spiral ganglion at 12.5 dpc when postmitotic ganglion cells are migrating away from the cochlear anlage. The efferents begin to form the intraganglionic spiral bundle by 14.5 dpc and the inner spiral bundle by 16.5 dpc in the basal turn. They have extensive collaterals among supporting cells of the greater epithelial ridge from 16.5 dpc onwards. Afferents and efferents in the basal turn of the cochlea extend through all three rows of outer hair cells by 18.5 dpc. Selective labeling of afferent fibers at 20.5 dpc (postnatal day 1) shows that although some afferents are still in early developmental stages, some type II spiral ganglion cells already extend for long distances along the outer hair cells, and some type I spiral ganglion cells end on a single inner hair cell. These data support previous evidence that in mice the early outgrowth of afferent and efferent fibers is essentially achieved by birth.

The development of vestibular connections in rat embryos in microgravity.

Existing experimental embryological data suggests that the vestibular system initially develops in a very rigid and genetically controlled manner. Nevertheless, gravity appears to be a critical factor in the normal development of the vestibular system that monitors position with respect to gravity (saccule and utricle). In fact several studies have shown that prenatal exposure to microgravity causes temporary deficits in gravity-dependent righting behaviors, and prolonged exposure to hypergravity from conception to weaning causes permanent deficits in gravity-dependent righting behaviors. Data on hypergravity and microgravity exposure suggest some changes in the otolith formation during development, in particular the size although these changes may actually vary with the species involved. In adults exposed to microgravity there is a change in the synaptic density in the optic sensory epithelia suggesting that some adaptation may occur there. However, effects have also been reported in the brainstem. Several studies have shown synaptic changes in the lateral vestibular nucleus and in the nodulus of the cerebellum after neonatal exposure to hypergravity. We report here that synaptogenesis in the medial vestibular nucleus is retarded in developing rat embryos that were exposed to microgravity from gestation days 9 to 19.

Electron microscopic differentiation of directly and transneuronally transported DiI and applications for studies of synaptogenesis.

The neuronal tracer DiI is a lipophilic dye which diffuses along the lipid bilayer of membranes and sometimes will move transcellularly. We used this tracer to study the development of olivocochlear synapses in the auditory system in rats and chickens by applying DiI directly to severed axons in the olivocochlear bundle. Observations with epi-fluorescent microscopy showed that DiI had labelled efferent axons directly and had labelled spiral ganglion cells and hair cells transneuronally. Ultrastructural analysis of photoconverted DiI tissue in rats revealed that transneuronal diffusion occurred when the plasma membrane of directly labelled axons made contact with the plasma membrane of their target structure. Directly and transneuronally labelled profiles can be distinguished easily at the electron microscopic level. In directly labelled profiles, all plasma, nuclear, endoplasmic reticulum, and outer mitochondrial membranes and cell cytoplasm are labelled leaving only the mitochondrial matrix unstained. However, in transneuronally labelled cells the endoplasmic, nuclear, and immature synaptic membranes are labelled but mitochondrial and non-synaptic plasma membranes are not labelled. This labelling pattern can be explained by diffusion through continuous membranes. These characteristics make DiI diffusion a powerful technique for identifying and studying early events in neuronal development and synapse formation.

Afferent projections to the ventromedial hypothalamic nucleus in a lizard, Gekko gecko.

Afferent projections to the ventromedial hypothalamic nucleus (VMH) of a lizard, Gekko gecko, were studied with wheat germ agglutinin conjugated to horseradish peroxidase. Results indicated that the shell and core areas of the VMH are largely innervated by different telencephalic nuclei but similar brain-stem nuclei. The common brainstem projections include the superior raphe, two isthmal populations (the ventral isthmal nucleus and parvocellular isthmal nucleus), and a dorsal thalamic projection from the posteroventral nucleus. Brainstem projections to the shell but not to the core of the VMH arise from the laterodorsal tegmental nucleus. Telencephalic projections to the VMH core originate from the ventrolateral septum, centromedial DVR, lateral amygdala, medial amygdala, interstitial amygdala, and ventral anterior amygdala. Telencephalic projections to the VMH shell come from the ventral pallidum, the anterior septal nucleus, the dorsal septal nucleus, the striatoamygdalar area, and the ventral posterior amygdala. These results, combined with connectional and topological information from other studies in amphibians, reptiles, and mammals, will be used to suggest homologies between a number of limbic areas, including several amygdalar nuclei.

Afferent projections to the lateral and dorsomedial hypothalamus in a lizard, Gekko gecko.

Afferent projections to the lateral hypothalamic area and dorsomedial hypothalamic nucleus of the lizard Gekko gecko were studied after applications of wheat germ agglutinin conjugated to horseradish peroxidase. Large applications into the hypothalamus labeled several telencephalic populations not observed after smaller injections. These included the rostrolateral area of the dorsal cortex, a sheet of cells deep to the caudal pole of the lateral cortex, the external amygdala, and part of the dorsal ventricular ridge. Other populations were labeled in the diencephalon, including the supraoptic nucleus and nucleus ovalis; in the medulla the medial reticular area was labeled. Injections into the lateral hypothalamic area labeled neurons in the rostrolateral dorsal cortex, anterior, lateral, and dorsal septal nuclei, the striatoamygdalar area, nucleus accumbens, vertical limb of the diagonal band, nucleus of the accessory olfactory tract, the interstitial, ventral anterior, and ventral posterior amygdalar nuclei, several hypothalamic nuclei, and the posteroventral thalamic nucleus. Labeled brainstem populations included the ventral tegmental area, torus semicircularis, parvocellular and ventral isthmal nuclei, superior raphe, and the solitary nucleus. Injections in the dorsomedial hypothalamic nucleus labeled neurons in the rostral and caudolateral poles of the dorsal cortex, anterior septal nucleus, horizontal limb of the diagonal band, nucleus of the anterior commissure, several hypothalamic areas, the lateral habenula, the posteroventral thalamic nucleus, and cells scattered around the dorsolateral anterior thalamic nuclei. Labeled brainstem populations included the torus semicircularis, ventral tegmental area, superior raphe, parvocellular and ventral isthmal nuclei, and the lateral dorsal tegmental nucleus. The results of these studies are compared with findings in amphibians and mammals.

The limbic system of tetrapods: a comparative analysis of cortical and amygdalar populations.

Recent studies of the limbic system of tetrapods have made data available that challenge some of the long-held tenets of forebrain evolution. Using the basic principle of parsimony--that the best hypotheses concerning homologies are those requiring the fewest number of evolutionary changes--we have reevaluated comparisons of tetrapod limbic systems. Given the current data, the following points appear to be justified: (1) the common ancestors of reptiles and mammals had a well-developed limbic system in which the basic subdivisions and connections of the amygdalar nuclei were established; (2) the ventral part of the lateral pallium in amphibians appears to be a single structure which corresponds to at least four areas in reptiles: centromedial DVR, ventral anterior amygdala, lateral amygdala, and part of the lateral cortex; (3) the medial pallium in amphibians appears to be homologous with the dorsal and medial cortices in reptiles and with the general and hippocampal cortices in mammals: (4) the cortical targets of the main olfactory bulb in reptiles and mammals appear to be homologous, and their common ancestor probably had a corresponding olfactory pallial field; (5) the targets of the accessory olfactory bulb in amphibians, reptiles, and mammals appear to be homologous, with the exception of nucleus sphericus in reptiles, which lacks an obvious homologue in non-reptiles.

Postnatal development and specification of the cat's visual corticotectal projection: efferents from the posteromedial lateral suprasylvian area.

The corticotectal projection in adult cats has a precise topographic and laminar organization. Yet this projection initially grows beyond these adult targets. To begin to understand how the growing cortical axons achieve this precision, the morphological development of axons growing from the posteromedial lateral suprasylvian area (PMLS) to the superior colliculus was studied by injecting the anterograde tracer biocytin into the PMLS of cats between postnatal day (P0) and adulthood. The labeling patterns showed that (1) axons grow independently towards the colliculus and (2) the first axons from the PMLS arrive in the colliculus by P1 and continue to arrive over several days. Labeled growth cones were seen within the colliculus up to P15. (3) After reaching the colliculus, the axons undergo several morphological changes. Initially, they are unbranched and beaded, then short side branches are formed and finally extensive arborizations appear. Comparing the timing of these events with results from electron microscopic and electrophysiological studies suggests that the appearance and increase in labeled axons with short side branches roughly coincides with the appearance and increase in number of synapses in the colliculus, whereas the elaboration of extensive arbors (and hence a corresponding increase in synapses) is well underway before visual cortical influences on the colliculus can be measured. Thick sinuous axons are also labeled during maturation, usually in areas of the colliculus where they would be considered exuberant and may represent degenerating axons. (4) A coarse topography develops as the axons grow into the colliculus and becomes more precise in the following weeks. Initially, some axons extend well beyond their correct terminal zone, growing into the contralateral colliculus, caudally into the inferior colliculus and reaching all laminae of the ipsilateral superior colliculus. Similar targeting 'errors' have been reported during the growth of retinotectal axons, suggesting that cortical, retinal and perhaps other sources as well, may use the same extracellular cues to establish an initial coarse topography within the colliculus.

Transient projections from the lateral geniculate to the posteromedial lateral suprasylvian visual cortex in kittens.

The postnatal maturation of the projection from the lateral geniculate nucleus to the posteromedial lateral suprasylvian visual cortex (PMLS) was studied with injections of fluorescent dyes into the PMLS at various postnatal ages. Labeled neurons projecting to the PMLS were present in all laminae of the ipsilateral lateral geniculate on the day of birth. However, there was a conspicuous change in the distribution of labeled geniculo-PMLS neurons by 11 days of age: now very few labeled neurons were present in lamina A, indicating a loss of geniculo-PMLS connections. The loss of connections began at the peripheral margins of lamina A and proceeded through other laminae toward laminae C1-3. By adulthood, labeled geniculo-PMLS neurons were largely confined to laminae C1-3; they were never observed in lamina A or A1 and were rarely observed in lamina C. To determine whether the lateral geniculate neurons survived after their projections to PMLS were lost, injections of fast blue were made at 1 or 2 days postnatally and the animals were allowed long postinjection survival times. Labeled neurons were found in all lateral geniculate laminae, thereby indicating that for many neurons the loss of connections could be attributed to a loss of their axon collaterals rather than to the death of the neurons themselves. After injections of fast blue into the PMLS and diamidino yellow dihydrochloride into area 17 shortly after birth, many double-labeled neurons were present in all laminae, indicating that they have collaterals to both targets. Thus, the survival of many of the geniculo-PMLS neurons contributing to the transient geniculo-PMLS projection seems to be due to sustaining collateral projections to area 17 or other cortical targets.

The organization of trigeminotectal and trigeminothalamic neurons in rodents: a double-labeling study with fluorescent dyes.

Retrogradely transported fluorescent dyes (fast blue and diamidino-dihydrochloride yellow) were used to compare the distributions of trigeminofugal neurons that project to the superior colliculus and/or the thalamus in three rodent species. The objective was to determine what the projection and collateralization patterns of these trigeminofugal pathways are and whether they are similar among different species. In each anesthetized animal, one dye was injected into the superior colliculus and the other into the topographically congruent area of the thalamus. Counts of the numbers of yellow, blue, and double-labeled neurons were made throughout the trigeminal complex: principalis, pars oralis, pars interpolaris, and pars caudalis. Trigeminothalamic projections were similar in each of the rodent species studied. The densest concentration of retrogradely labeled neurons was in principalis, with substantially fewer neurons in pars interpolaris, and fewer still in pars oralis and pars caudalis. These neurons were generally small and tended to have round or fusiform somata. A common pattern was also noted among the three species for trigeminotectal neurons. Most trigeminotectal projections originated from neurons in pars interpolaris, somewhat fewer from pars oralis, and the fewest from principalis and pars caudalis. These neurons tended to be the largest in each subdivision and were often multipolar. Following paired injections of the tracers, double-labeled neurons were scattered throughout the sensory trigeminal complex and had morphologies characteristic of single-labeled trigeminotectal neurons. Although comparatively few double-labeled neurons were observed in any species, most of those seen were restricted to the ventrolateral portion of pars interpolaris, a position that corresponds to the representation of the vibrissae. These data indicate that, regardless of the rodent species, the vast majority of labeled trigeminal neurons project either to the superior colliculus or the thalamus, but not to both targets. This might be expected on the basis of the very different behavioral roles these structures play. On the other hand, a subpopulation of trigeminal neurons exists (mainly in pars interpolaris) that does project to both the superior colliculus and the thalamus, perhaps because both structures require some of the same somatosensory information to perform their behavioral functions.

Telencephalic connections in lizards. I. Projections to cortex.

The afferent connections to five cortical regions in two distantly related species of lizards (Gekko gecko and Iguana iguana) were studied by means of retrograde transport of horseradish peroxidase conjugated to wheat germ agglutinin. Each of the five cortical regions is characterized by a specific pattern of projections from telencephalic, thalamic, hypothalamic, and brainstem regions. Subdivisions within the five cortical regions also receive different patterns of projections. The thalamo-cortical projections are as follows: The small-celled mediodorsal cortex receives a projection from nucleus dorsolateralis anterior pars magnocellularis. The large-celled mediodorsal cortex receives projections from nucleus dorsolateralis anterior pars parvicellularis and pars magnocellularis. The dorsal cortex receives a projection from nucleus dorsolateralis anterior pars parvicellularis. The lateral cortex receives a projection from nucleus dorsolateralis anterior pars magnocellularis. The pallial thickening receives projections from nucleus dorsomedialis and nucleus intercalatus. The latter nucleus receives a direct retinal projection. Thus, the pallial thickening is the recipient of a retino-thalamocortical projection. To date, comparisons of data from experimental studies have suggested that the cortical regions in lizards and turtles may be organized differently. However, the results of the present study suggest that the organization of cortical regions among reptiles is more similar than previously realized.

Telencephalic connections in lizards. II. Projections to anterior dorsal ventricular ridge.

Three distinct cytoarchitectonic regions were identified within the anterior dorsal ventricular ridge (ADVR) of two species of lizards, Gekko gecko and Iguana iguana. These regions have been named according to their general topographical positions: medial area, caudolateral area, and rostrolateral area. Injections of horseradish peroxidase throughout the ADVR demonstrated that each of the three areas of the ADVR receives projections from specific thalamic nuclei which are associated with specific sensory modalities. The medial area receives an auditory thalamic projection from nucleus medialis. The caudolateral area receives thalamic projections from nucleus medialis posterior and nucleus posterocentralis. The latter two nuclei were shown to receive projections from the spinal cord and, therefore, are presumed to be associated with body somatosensory information. The rostrolateral area receives a thalamic projection from nucleus rotundus, which receives visual information. In addition, the mesencephalic tegmentum and the thalamic nucleus dorsomedialis project to the entire ADVR. The latter projection is similar to the diffuse cortical projections of the intralaminar thalamic nuclei in mammals. These findings support previous suggestions that the ADVR is comparable to sensory regions of the mammalian neocortex.

The sources of supraspinal afferents to the spinal cord in a variety of limbed reptiles. I. Reticulospinal systems.

Horseradish peroxidase was injected into various levels of the spinal cord of turtles (Pseudemys and Chrysemys), lizards (Tupinambis, Iquana, Gekko, Sauromelus, and Gerrhonotus), and a crocodilian (Caiman). The results suggest that brainstem reticulospinal projections in limbed reptiles rival mammalian reticulospinal systems in complexity. The reptilian myelencephalic reticular formation can be divided into four distinct reticulospinal nuclei. Reticularis inferior pars dorsalis (RID) contains multipolar neurons which project bilaterally to the spinal cord. Reticularis inferior pars ventralis (RIV), which is only found in lizards and crocodilians, contains fusiform neurons with horizontally running dendrites and it projects ipsilaterally to the spinal cord. Reticularis ventrolateralis (RVL), which is found only in field lizards, contains triangular neurons whose dendrites parallel the ventrolateral edge of the brainstem and it projects ipsilaterally to the spinal cord. The myelencephalic raphe (RaI) varies considerably. RaI of turtles contains large reticulospinal neurons which form a continuous population with more laterally situated RID cells. RaI of lizards contains a few small reticulospinal neurons. RaI of the crocodilian Caiman contains giant reticulospinal neurons with laterally directed dendrites. The caudal metencephalic reticular formation of reptiles can be divided into two distinct reticulospinal nuclei. Reticularis medius (RM) contains large neurons with long, ventrally directed dendrites; it projects ipsilaterally to the spinal cord. Reticularis medius pars lateralis (RML) contains small neurons with laterally directed dendrites; it projects contralaterally to the spinal cord. The rostral mesencephalic and caudal mesencephalic reticular formation of reptiles can be divided into three distinct reticulospinal nuclei. Reticularis superior pars medialis (RSM) consists mostly of small, spindle-shaped neurons which project bilaterally to the spinal cord. In the lizard Tupinambis, however, large multipolar, ipsilaterally projecting neurons are occasionally seen in RSM. Reticularis superior pars lateralis (RSL) contains large, ipsilaterally projecting neurons with long, ventrolaterally directed dendrites. SRL in lizards can be divided into a dorsomedial portion, which projects ipsilaterally to the spinal cord, and a ventrolateral portion which projects contralaterally. The locus ceruleus-subceruleus field (LC-SC) contains small spindle-shaped neurons which project bilaterally to the spinal cord. Labelled reticulospinal neurons were also observed in the rostral metencephalic raphe (RaS) of the turtle brainstem. These cells are small, spindle-shaped neurons which resemble the small cells of the adjacent RSM field.

Nucleus laminaris of the torus semicircularis: projection to spinal cord in reptiles.

Neurons in nucleus laminaris of the torus semicircularis were retrogradely labeled following application of horseradish peroxidase (HRP) to the cervical spinal cord in two lizards (Gekko gecko and Iguana iguana) and a turtle (Pseudemys scripta). Different patterns of cell labeling were seen among the species studied and may be related to the distinctive differences in head and body movements seen in these animals during defensive, aggressive and social behaviors.