The structure and development of avian lumbosacral specializations of the vertebral canal and the spinal cord with special reference to a possible function as a sense organ of equilibrium.

The avian lumbosacral vertebral column and spinal cord show a number of specializations which have recently been interpreted as a sense organ of equilibrium. This sense organ is thought to support balanced walking on the ground. Although most of the peculiar structures have been described previously, there was a need to reevaluate the specializations with regard to the possible function as a sense organ. Specializations were studied in detail in the adult pigeon. The development of the system was studied both in the pigeon (semiprecocial at hatching) and in the chicken (precocial). Specializations in the vertebral canal consist of a considerable enlargement, which is not due to an increase in the size of the spinal nervous tissue, but to a large glycogen body embedded in a dorsal rhomboid sinus. The dorsal wall of the vertebral canal shows segmented bilateral dorsal grooves, which are covered by the meninges towards the lumen of the vertebral canal leaving openings in the midline and laterally. This results in a system of lumbosacral canals which look and may function similar to the semicircular canals in the inner ear. Laterally these canals open above ventrolateral protrusions or accessory lobes of the spinal cord which contain neurons. There are large subarachnoidal cerebrospinal fluid spaces, lateral and ventral to the accessory lobes. Movement of this fluid is thought to stimulate the lobes mechanically. As to the development of avian lumbosacral specializations, main attention was given to the organization of the lobes and the adjacent fluid spaces including the dorsal canals. In the pigeon the system is far from being adult-like at hatching but maturates rapidly after hatching. In the chicken the system looks already adult-like at hatching. The implications derived from the structural findings are discussed with regard to a possible function of the lumbosacral specializations as a sense organ of equilibrium. The adult-like organization in the newly hatched chickens, which walk around immediately after hatching, supports the assumed function as a sense organ involved in the control of locomotion on the ground.

Embryonic development of choline acetyltransferase and nitric oxide synthase in the spinal cord of pigeons and chickens with special reference to the superficial dorsal horn.

The superficial dorsal horn of birds as well as mammals contains both cholinergic and nitrergic neuronal structures as evident from the presence of the synthesizing enzymes such as choline acetyltransferase and nitric oxide synthase, which is an NADPH diaphorase. In the rat, both systems develop only postnatally. Rats are altricial at birth whereas pigeons and chickens are semiprecocial or precocial, respectively, at the time of hatching. Immunocytochemical studies of choline acetyltransferase and nitric oxide synthase in the developing avian spinal cord (starting with embryonic day 12 of 18 in the pigeon and 14 of 21 in the chicken) showed that both systems are well developed in the superficial dorsal horn at the time of hatching in both avian species. In the pigeon, choline acetyltransferase-positive superficial dorsal horn neurons appear only on the day of hatching (E18), whereas nitric oxide synthase-positive neurons can be first detected at stage E14. In the chicken, nitric oxide synthase-positive neurons are present already at stage E14, whereas choline acetyltransferase-positive neurons appear at stage E20. Autonomic and somatic motor neurons show adult-like choline acetyltransferase-immunoreactivity and/or nitric oxide synthase-immunoreactivity at the earliest stages investigated. It is concluded that the stage of maturation at birth or hatching plays an important role in the development of superficial dorsal horn cholinergic and nitrergic systems.

Distribution of choline acetyltransferase and NADPH diaphorase in the spinal cord of the pigeon.

Acetylcholine is the neurotransmitter of somatic and autonomic motor systems of the spinal cord. However, there are also intrinsic cholinergic systems which have modulatory functions. Modulatory functions have also been assigned to nitric oxide (NO). Acetylcholine is synthesized by choline acetyltransferase and NO by nitric oxide synthase, which is a NADPH diaphorase. The distribution of both enzymes in the mammalian spinal cord is well known. However, there is a lack of comparative data in avian species. Therefore, the distribution of both enzymes in the spinal cord of the pigeon was studied using histochemical and immunohistochemical methods. Aside from somatic motor neurons and autonomic preganglionic neurons choline acetyltransferase-immunoreactivity was found throughout the spinal cord in lamina III of the superficial dorsal horn and near the central canal. The location of choline acetyltransferase-positive preganglionic neurons in the centrally located column of Terni and the lack of an intermediolateral column typical of the mammalian spinal cord can be confirmed. In lumbosacral segments the axons of centrally located cholinergic neurons crossed to the contralateral side to form a tract in the ventral funiculus, which then innervates the contralateral grey substance. A dense band of NADPH diaphorase staining was found in lamina II and in centrally located neurons of all segments. Part of the centrally located neurons double-labelled for choline acetyltransferase and NADPH diaphorase. In contrast to mammals, preganglionic neurons labelled only weakly for NADPH diaphorase. Altogether, despite the divergent evolution of both classes of vertebrate intrinsic modulatory choline acetyltransferase and NADPH diaphorase systems of birds seem to be largely similar to those of the mammalian spinal cord.

Histochemical and immunocytochemical investigations of the marginal nuclei in the spinal cord of pigeons (Columba livia).

In birds there are segmentally organized marginal nuclei at the lateral or ventrolateral border of the spinal cord. In most regions of the spinal cord these nuclei are within the outline of the cord. However, in the lumbosacral region they form accessory lobes protruding into the vertebral canal. Histochemical and immunocytochemical investigations were performed to study the neurochemical features of the marginal nuclei of the pigeon. Despite histological differences (only accessory lobe neurons are embedded in glia-derived glycogen cells), there was no difference in the chemical neuroanatomy of the two types of marginal nuclei. These nuclei contained cholinergic neurons and there was also evidence for a cholinergic innervation. NADPH-diaphorase activity, which is considered to indicate nitric oxide synthesis, was faint in marginal neurons. No serotonin immunoreactivity was found. However, all neurons showed immunoreactivity to glutamate and glycine, and some were immunoreactive to gamma-aminobutyric acid (GABA). A GABAergic innervation of non-GABAergic neurons could also be demonstrated. The lack of difference in the chemical neuroanatomical features between cervical marginal nuclei and lumbosacral accessory lobes suggests a similar origin of all marginal neurons. A comparison with the chemical neuroanatomy of marginal neurons in other vertebrates shows both similarities and differences.

Spinocerebellar projections in the pigeon with special reference to the neck region of the body.

Neck sensory information is important for control of head and body movements in all vertebrates. Neuroanatomic tracing methods were used to study the pathways of neck afferent systems. Both the projection of primary afferent fibers and of secondary afferent pathways to brainstem and cerebellum were investigated with the anterograde transport of dextran amines as tracers (biotinylated dextran amine and tetramethyl rhodamine dextran amine). For comparison, the projections of spinocerebellar systems of wing and leg were studied also. Complementary experiments using retrograde tracers (Fast Blue, tetramethyl rhodamine dextran amine, rhodamine isothiocyanate) injected into the cerebellum served to corroborate the results of the anterograde tracing experiments. Primary neck afferent fibers terminated in the spinal gray substance with dense terminal fields in laminae I to V of the dorsal horn and lamina IX of the ventral horn as well as in the marginal nuclei located at the lateral border of the spinal cord. In the brainstem, dense terminal fields were seen in deep layers of the medullary dorsal horn, in the external cuneate nucleus, and in group x. Secondary neck afferents arising from ventral horn cells showed a significant projection to the descending and medial vestibular nuclei and to the medial cerebellar nucleus. Terminals were found both in the anterior and the posterior cerebellum. A quantitative evaluation disclosed that most terminals of neck afferents distributed in lobules II-IV of the anterior cerebellum and lobule IX of the posterior cerebellum. With injections aimed at spinocerebellar neurons located into the cervical and lumbosacral enlargements, no projections were found in the vestibular or deep cerebellar nuclei. Projections from the cervical enlargement were concentrated in lobules III-V and those from the lumbosacral enlargement in lobules III-VI. This points to a rostrocaudal somatotopic representation of neck, wing, and leg in the anterior cerebellum. The results of the retrograde tracing experiments support such a somatotopic organization.

Spinomedullary pathways in the pigeon (Columba livia): differential involvement of lamina I cells.

The lamina I (marginal zone) of the spinal cord dorsal horn is an important site for pain processing. In mammals, lamina I neurons have been shown to constitute a heterogeneous population made up of four morphological groups with particular neurochemical nature, supraspinal connection patterns, and nociceptive response properties. In order to obtain a comparative view of the mechanisms of nociceptive processing, the analysis of the structural morphology and supraspinal connectivity of lamina I neurons was, in this study, extended to the avian family. Cholera toxin subunit B (CTb) was injected in the nucleus tractus solitarius (NTS), nucleus centralis medullae pars dorsalis (Cnd), and the dorsolateral portion of the nucleus reticularis lateralis (RLlat) of the pigeon (Columba livia), areas equivalent to the rat caudal medulla oblongata lamina I targets, which have been shown to receive differential projections from all cell groups present in lamina I of mammals. In the pigeon, lamina I cells project to the three medullary regions and present the same morphology of spinomedullary lamina I cells of mammals: the spinal-NTS and the spinal-RLlat pathways originated from fusiform, pyramidal, and flattened neurons, and the spinal-Cnd pathway from multipolar, pyramidal, and flattened neurons. Furthermore, the relative participation of each lamina I cell type in each pathway was found to be similar to that previously observed in the rat. The observed similarities on the anatomical organization of lamina I neurons in mammalian and avian species can be taken as a phylogenetic indication of the importance of the nociceptive circuitry centered in lamina I.

Behavioral evidence of the role of lumbosacral anatomical specializations in pigeons in maintaining balance during terrestrial locomotion.

In birds there are anatomical specializations in the lumbosacral vertebrae and spinal cord. These include segmentally organized bony canals which are related to accessory lobes of the spinal cord. Both structures are connected by cerebrospinal fluid. To test whether these specializations function as a sense organ of equilibrium the effect of opening the fluid space was studied in pigeons. Locomotory behaviors on the ground (landing on a perch, keeping balance on a rotating perch, walking) but not flight were significantly impaired after lesion. These results support the assumption that the lumbosacral specializations are involved in the control of locomotion on the ground.

Fine structural evidence of mechanoreception in spinal lumbosacral accessory lobes of pigeons.

In the lumbosacral spinal cord of birds there are accessory lobes which protrude into the vertebral canal. The accessory lobes consist of multipolar neurons and glia-derived glycogen cells. It has been suggested that these lobes function as a sense organ of equilibrium. Therefore the lobes were studied ultrastructurally to look for possible mechanoreceptive structures. Extracellular lacunae extend from the periphery deep into each lobe. The dendrites of neurons ramify into the lacunae where they issue finger-like processes which do not contact any other cells and which are not contacted by boutons. Since finger-like processes are typical of peripheral and central mechanoreceptive neurons it is concluded that the observed processes indicate a mechanoreceptive function of the lobe neurons.

Fibre composition in the interosseous nerve of the pigeon.

The interosseous nerve of birds innervates a string of Herbst corpuscles located near the interosseous membrane between the tibia and fibula. Fibre composition of this nerve was assessed including both myelinated and unmyelinated axons. The diameter of the whole nerve is approximately 100 microm. Complete data were obtained for 3 nerves. The mean total number of myelinated fibres and unmyelinated axons was 2872 +/- 53. The mean number of myelinated fibres was 280 +/- 20 and that for unmyelinated axons was 2600 +/- 47. There was a broad distribution of diameters for myelinated fibres ranging from approximately 2 microm to 10 microm with a distinct peak at approximately 3-5 microm and a less prominent second peak at 6-8 microm. Similarly, myelin sheath thickness distribution showed 2 peaks, one at 0.6-0.8 microm and another at 1.4-1.6 microm. It is suggested that the group represented by the second peak innervates the Herbst corpuscles. The group of smaller myelinated fibres and the unmyelinated axons are assumed to innervate other types of receptors, some of which may be nociceptors.

Specializations in the lumbosacral spinal cord of birds: morphological and behavioural evidence for a sense of equilibrium.

Birds have a variety of long known anatomical specializations both in the vertebrae and in the spinal cord of lumbosacral segments. In the present investigation additional morphological specializations are described for the pigeon. These consist of segmentally organized semicircular canal-like structures (lumbosacral canals) which together with specializations in the meninges of the spinal cord form a large liquor space above accessory lobes attached to the spinal cord. The whole system is thought to function as a sense of equilibrium. The neurons in the lobes are assumed to be sensory neurons which are stimulated by the inertia of the fluid during movements of the body. Such a function is supported by lesion experiments: opening of the fluid space was followed by severe disturbances of landing and walking behavior.

Response characteristics of cerebellar nuclear cells in the pigeon.

In birds, the output system of the cerebellum, the cerebellar nuclei, has not yet been studied electrophysiologically. Recordings from nuclear cells during electrical stimulation of the radial nerve revealed a uniform type of response consisting of an initial inhibition followed by a clear cut excitation. Responses with an initial excitation were rare. Response patterns and latencies suggest an input from Purkinje cells of the cerebellar cortex and a lack of collateral input from spinocerebellar pathways. This points to a fundamental difference from cerebellar nuclear cells in mammals, in which collateral input provides a prominent excitatory response under comparable experimental conditions.

Size of somatosensory cortex and of somatosensory thalamic nuclei of the naturally blind mole rat, Spalax ehrenbergi.

The hypothesis that the somatosensory system in the naturally blind subterranean rodent Spalax ehrenbergi (= mole rat) is enlarged was tested by measuring the volume of somatosensory cortex and somatosensory thalamic nuclei (Nuclei ventrales posteromedialis and posterolateralis). Electrophysiology and tracing were used to identify and delineate these areas. On average the somatosensory cortex is 1.7 times larger and the thalamic nuclei are 1.3 times larger in the blind mole rat than in the sighted laboratory rat if different body weights are taken into consideration. This confirms the demands of a life underground where it seems touch would replace vision. The data reveal a remarkable brain plasticity among mammals under natural conditions.

Projections of the marginal nuclei in the spinal cord of the pigeon.

In the avian spinal cord, there are several groups of neurons lying outside the central gray substance. The most conspicuous ones lie at the very margin of the ventrolateral cord. In the lumbosacral spinal cord, these marginal nuclei protrude into the vertebral canal to form accessory lobes. The projections of these marginal nuclei were studied in the pigeon by neuroanatomical tracing methods. Anterograde transport of tracer injected into the lumbosacral accessory lobes showed that these neurons project to the contralateral medial ventral gray and to paragriseal cells located in the contralateral ventral and lateral white matter of lumbosacral segments. Double-labeling experiments disclosed that lumbosacral paragriseal cells projecting to the cerebellum are contacted by accessory lobe axon terminals. The projection of cervical marginal nuclei was studied with retrograde transport of tracers applied to the spinal tracts in the lateral funiculus. Retrogradely labeled cells were found in contralateral marginal nuclei of both rostral and caudal segments. All marginal nuclei have an ascending and a descending projection spanning about five segments each. The possible role of marginal nuclei in sensorimotor circuits is discussed.

Electrophysiological investigations of the somatosensory thalamus of the pigeon.

Somatosensory areas in the thalamus of the pigeon were studied electrophysiologically at the single-unit level. Of 459 units, 429 responded to somatosensory stimuli. Of these, 394 units responded specifically to somatosensory stimuli, whereas 8 responded in addition to visual stimuli and 27 to auditory stimuli also (bimodal neurons). Seven units were exclusively driven by visual stimuli and 23 units by auditory stimuli. Recording sites included nucleus dorsalis intermedius ventralis anterior (DIVA) and nucleus dorsolateralis posterior (DLP). The number of bimodal or visual and auditory neurons was much larger in DLP (40%) than in DIVA (7%). This points to a specific somatosensory function of DIVA. There was only a poor somatotopic organization in both nuclei. However, in vertical penetrations, wing representation was usually dorsal to leg representation. Distal parts of the limbs had smaller receptive fields (RFs) than proximal parts, and the smallest RFs were found on the toes, which seem to be represented in most detail.

Spinal distribution and brainstem projection of lamina I neurons in the pigeon.

Lamina I neurons of the spinal dorsal horn serve nociception both in mammals and in birds. The projection of these neurons to the brain is largely unknown in birds. Injections of retrogradely transported fluorescent tracers into various brainstem nuclei showed that these neurons, which are distributed throughout the spinal cord, heavily project to the nucleus of the solitary tract and the parabrachial area but not to the hypothalamus. Injections into the nucleus of the solitary tract revealed a group of neurons located in Lissauer's tract of thoracic segments. These results point to a functional role of spinal lamina I neurons in avian visceronociception.

Central projections of primary afferents from the interosseous nerve in the pigeon.

The interosseous nerve in the pigeon's leg innervates a string of Herbst corpuscles. Because Herbst corpuscles are vibration-sensitive, this study, using neuronal tracing methods, was expected to show the central representation of vibration sense. After application of a mixture of free and lectin-conjugated horseradish peroxidase to the interosseous nerve, labeled cell bodies of sensory and postganglionic neurons were mainly located in the dorsal root ganglia and paravertebral sympathetic ganglia L3/L4. In spinal segments L3/L4 fibers and terminals were mainly distributed at the lateral border of the head of the dorsal horn. In more cranial or caudal segments terminal fields were at intermediate parts of laminae I/II and laminae IV/V. Some labeled fibers entered the dorsal horn from medial to terminate in lamina IV. Primary afferents of the interosseous nerve projected directly to the gracile nucleus in the brainstem and distributed all along its rostrocaudal extent. Because the main terminal fields in the spinal cord are typical for the projection of small afferent fibers, vibration information seems to reach the brainstem via the dorsal column primary afferents.

Functional anatomy of the thalamus in the blind mole rat Spalax ehrenbergi: an architectonic and electrophysiologically controlled tracing study.

The occipital cortex of the naturally blind mole rat, Spalax ehrenbergi, is occupied by an area of somatosensory representation. To date, no visual cortex has been identified electrophysiologically. In order to determine whether there are corresponding modifications in the thalamus, thalamocortical connections were studied with neuroanatomical tracing methods. Three different fluorescent tracers were injected under electrophysiological control into distinct cortical areas. Injections into the somatosensory head/face and hindlimb/trunk areas of representation revealed a posteromedial ventral nucleus and a posterolateral ventral nucleus, respectively. Additional somatotopic labeling was found in an area dorsomedial to the two ventral nuclei. This structure may be equivalent to the posterior nuclear complex in the laboratory rat. Injections into the auditory cortex of the mole rat resulted in labeling of the medial geniculate body. In contrast to the situation in the laboratory rat, in which a prominent dorsolateral geniculate body and a ventrolateral geniculate body assume dorsolateral positions, the somatosensory thalamus of the mole rat almost reaches the dorsolateral surface. This finding is corroborated by the results of the architectonic study, which failed to reveal a differentiated lateral geniculate body. Our observations suggest that the thalamocortical visual system in the mole rat is minute, whereas the somatosensory system is expanded. This situation fits the mode of life of this subterranean animal, for which touch is more important than vision.

Sensorimotor aspects of flight control in birds: specializations in the spinal cord.

Avian bipedal locomotion and flight suggest specializations in sensorimotor integration as compared to quadrupedal vertebrates. Specializations at the level of the spinal cord were studied with neuroanatomical tracing experiments in pigeons. Bird-specific connections were found in the exteroreceptive system (receptors associated with feathers) and in the proprioreceptive system (spinocerebellar pathways). These specializations seem to be related to flight control. Neurons of the marginal nuclei (accessory lobes) in the lumbosacral cord, which are unique to birds, have propriospinal connections mainly to the contralateral ventral cord. The functional role of these nuclei remains enigmatic.

Projection of wing nerves to spinal cord and brain stem of the pigeon as studied by transganglionic transport of Fast Blue.

To see whether there is a topographic organization of forelimb nerves in the CNS of birds, the termination pattern of afferents from wing nerves of the pigeon in the cervical spinal cord and the brain stem was determined by the transganglionic transport of Fast Blue and HRP. Fast Blue turned out to be a very sensitive and nonselective tracer with a wider distribution of terminal labeling than with HRP. Thus, Fast Blue is a useful marker for complete mapping of the terminal fields of peripheral nerves. Despite considerable overlap of the terminal fields of individual nerves the areas of densest labeling were somatotopically organized in the spinal dorsal horn. This organization is very similar to that described for the mammalian forelimb. In the rostral cervical segments all nerves have a projection field in ventromedial parts of the dorsal horn but there is no topographic organization. In the medulla terminal fields appear in the dorsal column nuclei including the external cuneate nucleus and group x near the descending vestibular nucleus. In sharp contrast to mammalian species there is no topographic representation of individual wing nerves in these brain stem areas.

Electrophysiological mapping of body representation in the cortex of the blind mole rat.

The cortex of the blind mole rat (Spalax ehrenbergi) was explored for somatosensory responses with special reference to an extension into the occipital cortex which serves vision in sighted mammals. Head and body representation was similar as in other rodents or mammals. However, the somatosensory area extended far into the occipital cortex. No responses to auditory or visual stimulation were found caudal to the somatosensory area. However, auditory responses were recorded in an area lateral to and slightly caudal to the head representation. It is concluded that in this naturally blind animal the area normally occupied by the visual cortex serves somatosensory function.