Glutamic acid decarboxylase immunoreactivity in the olfactory bulb of a reptile.

The objective is to determine the distribution of glutamic acid decarboxylase (GAD) in the olfactory bulb of a crocodilian, Caiman crocodilus . Avidin-biotin immunohistochemical methodology using a polyclonal antibody to GAD raised in sheep was employed. The following controls were used: substitution of the primary antibody with preimmune sheep serum at concentrations equal to that of the primary antibody; omission of the primary antibody; and omission of the primary antibody and biotinylated rabbit antisheep immunoglobulin. No GAD (+) cells were observed in the control sections. Based on cell and fiber staining, the layering and neuronal organization of the olfactory bulb in Caiman were similar to other vertebrates, including other reptiles. The following elements were GAD (+): granule cells, certain neurons in the outer plexiform layer, periglomerular neurons, and the glomeruli themselves. GAD (+) puncta were present throughout the olfactory bulb. In conclusion, these results in Caiman were similar, in part, to comparable studies in mammals and birds. Taken together, these data indicate that crocodiles not only have a similar pattern of layers that other amniotes possess but also that the immunocytochemical signatures of certain elements of the olfactory bulb are likewise shared.

Nuclei and Tracts in the Telencephalon of Crocodiles: Identification and Characterization Using an Organizational Scheme Applicable to Other Reptiles.

The telencephalon of reptiles has been suggested to be the key to understanding the evolution of the forebrain. Nevertheless, a meaningful framework to organize the telencephalon in any reptile has, with rare exception, yet to be presented. To address this gap in knowledge, the telencephalon was investigated in two species of crocodiles. A variety of morphological stains were used to examine tissue in transverse, horizontal, and sagittal planes of sections. Besides providing a description of individual nuclei, brain parts were organized based on two features. One was related to two fixed, internal structures: the lateral ventricle and the dorsal medullary lamina. The other was the alignment of neurons into either layers, cortex, or not, nucleus. Viewed from this perspective, all structures, with limited exceptions, could be accurately placed within the telencephalon regardless of the plane of section. Furthermore, this framework can be applied to other reptiles. A further extension of this scheme suggests that all structures in the telencephalon could be grouped into one of two categories: pallial or basal.

Interconnections between the dorsal thalamus and the basal nuclei in a reptile.

Reciprocal connections between the thalamus and the cortex are one of the most characteristic features of forebrain organization in mammals. To date, this circuit has been documented only in turtles. However, reptiles, including turtles, have an additional path from the dorsal thalamus to the telencephalon. This terminates in a pallial structure known as the dorsal ventricular ridge. Yet, no reciprocal connection from the dorsal ventricular ridge to thalamic nuclei has been uncovered. Since axons from the thalamus pass through the basal nuclei on route to the dorsal ventricular ridge, the basal nuclei might be a source of reciprocal connections. Accordingly, the location and distribution of neurons after retrograde tracer placement into the dorsal thalamus were examined. Retrogradely labeled neurons in the basal nuclei were indeed found. One possibility to explain this observation is that connections with the dorsal ventricular ridge are present during development but later pruned during embryogenesis.

Nuclei and tracts in the thalamus of crocodiles.

The thalamus is one of the most important divisions of the forebrain because it serves as the major hub for transmission of information between the brainstem and telencephalon. While many studies have investigated the thalamus in mammals, comparable analyses in reptiles are incomplete. To fill this gap in knowledge, the thalamus was investigated in crocodiles using a variety of morphological techniques. The thalamus consists of two parts: a dorsal and a ventral division. The dorsal thalamus was defined by its projections to the telencephalon, whereas the ventral thalamus lacked this circuit. The complement of nuclei in each part of the thalamus was identified and characterized. Alar and basal components of both the dorsal and ventral thalamus were distinguished. Although some alar-derived nuclei in the dorsal thalamus shared certain features, no grouping could account for all of the known nuclei. However, immunohistochemical observations suggested a subdivision of alar-derived ventral thalamic nuclei. In view of this, a different approach to the organization of the dorsal thalamus should be considered. Development of the dorsal thalamus is suggested to be one way to provide a fresh perspective on its organization.

Nuclei and tracts in the epithalamus of crocodiles.

The epithalamus, an area of the dorsal diencephalon found in all vertebrates, consists of the habenula, the subhabenular nuclei, and associated tracts. The habenula is itself divisible into two parts-a medial and a lateral nucleus differing in their inputs, outputs, and cellular morphology. The medial component is related to the limbic system and serotonergic raphe, while the lateral nucleus is more interconnected with the basal ganglia and midbrain dopamine systems. These findings, which come from experiments mainly done on mammals, serve as a basis for comparison with other vertebrates. However, similar studies in other amniotes, such as reptiles, are few. To fill this gap in knowledge, two species of crocodiles were examined utilizing a variety of histological methods in various planes of section. The following results were obtained. First, the habenula was divided into medial and lateral parts based on its cytoarchitecture. Neurons in the medial habenula were small, were closely packed, and had a limited dendritic arbor characterized by unusual distal dendritic appendages, whereas neurons in the lateral habenula were larger, were more loosely packed, and had longer dendritic processes that were commonly beaded. Second, the stria medullaris, the major input to the habenula, was identified by its immunoreactivity to parvalbumin. Third, the fasciculus retroflexus (habenulointerpeduncular tract), the primary output of the habenula, was visualized by staining with acetylcholinesterase. Fourth, nuclei associated with the habenula, the subhabenular nuclei, have been identified and characterized. These features provide a means to recognize the major nuclei and tracts in the epithalamus in crocodiles and are likely applicable to other reptiles.

A different framework to classify preoptic and hypothalamic nuclei in reptiles.

The preoptic area and the hypothalamus are inextricably linked. Together, they represent an area of the forebrain that is essential for survival of the species. Observations in mammals have suggested a classification of these structures into four rostrocaudal areas and three mediolateral zones. Two species of crocodiles were investigated to determine if this scheme or a modification of it could be applied to these reptiles. The resulting classification identified three rostrocaudal areas based on their respective relationship to the ventricular system: preoptic, anterior, and tuberal and four mediolateral zones: ependyma, periventricular, medial, and lateral. This scheme avoided the cumbersome and complicated nomenclature that has traditionally been used for morphologic studies of these areas in other reptiles, including crocodiles. The present classification is simple, straightforward, and readily applicable to other reptiles.

Evolution of Local Circuit Neurons in Two Sensory Thalamic Nuclei in Amniotes.

Local circuit neurons are present in the thalamus of all vertebrates where they are considered inhibitory. They play an important role in computation and influence the transmission of information from the thalamus to the telencephalon. In mammals, the percentage of local circuit neurons in the dorsal lateral geniculate nucleus remains relatively constant across a variety of species. In contrast, the numbers of local circuit neurons in the ventral division of the medial geniculate body in mammals vary significantly depending on the species examined. To explain these observations, the numbers of local circuit neurons were investigated by reviewing the literature on this subject in these two nuclei in mammals and their respective homologs in sauropsids and by providing additional data on a crocodilian. Local circuit neurons are present in the dorsal geniculate nucleus of sauropsids just as is the case for this nucleus in mammals. However, sauropsids lack local circuits neurons in the auditory thalamic nuclei homologous to the ventral division of the medial geniculate body. A cladistic analysis of these results suggests that differences in the numbers of local circuit neurons in the dorsal lateral geniculate nucleus of amniotes reflect an elaboration of these local circuit neurons as a result of evolution from a common ancestor. In contrast, the numbers of local circuit neurons in the ventral division of the medial geniculate body changed independently in several mammalian lineages.

A rare case of partial skull and brain duplication in a hatchling Alligator mississippiensis.

Errors in development occur in all vertebrates. When severe, these anomalies are lethal and frequently escape attention. In rare cases, animals with profound malformations are born and can provide a glimpse into structures and their respective function that would otherwise go unnoticed. A rare abnormality in a hatchling Alligator mississippiensis is described in which duplication of the skull, face, and brain was incomplete. The rostral skull, face, and associated forebrain, including the olfactory apparatus, were duplicated. However, the caudal skull and brainstem were not. These observations were made with advanced imaging using both computed tomography and magnetic resonance coupled with gross brain dissections. These abnormal features emphasize the complex and intertwined relationship between the development of the brain, face, and skull which are influenced by certain signaling molecules, possible gene mutation(s), and potential environmental factors.

Nuclei and tracts in the pretectum and associated tegmentum of crocodiles.

In all vertebrates, the pretectum and associated tegmentum arise from prosomere 1, but the adult derivatives of these embryonic regions are not well defined in reptiles-especially in crocodiles, the reptilian group most closely related to birds. Despite its importance in vision and visuomotor behavior, descriptions of the pretectum in crocodiles are brief and photographs are lacking. To fill this gap in knowledge, the pretectum and associated tegmentum were examined in two crocodilians, Caiman crocodilus and Alligator mississippiensis, using a variety of histological stains in all three traditional planes of section. These observations were compared with similar studies in other reptiles and birds. These comparisons were hampered by differences in nomenclature and limited data. Nevertheless, pretectal nuclei in receipt of retinal input in crocodiles, other reptiles, and birds were the most easily identified when compared with the present analysis. Despite identifying the traditional nuclei comprising the pretectum of crocodiles, other areas remain to be characterized. Nevertheless, knowledge gained from this description will aid further investigations of this brain region in crocodiles and other reptiles as well as provide a reference for developmental studies in crocodiles.

Do crocodiles have a zona incerta?

In mammals, the zona incerta is thought to be involved in a number of behaviors: visceral activity, arousal, attention, and posture and locomotion. These diverse and complex features suggested that the zona incerta functions as a global or integrative node. Nevertheless, despite multiple investigations into its anatomy, physiology, and behavior in a variety of mammals, no specific character identifies the zona incerta besides its appearance in fiber-stained material and its relationship to surrounding structures. One such structure is the thalamic reticular nucleus whose caudal pole often contains some intermingled cells of the zona incerta. In crocodilians, the entopeduncular nucleus (ep) abuts the caudal pole of the thalamic reticular nucleus and displays different immunohistochemical properties and soma size when compared with neurons in the thalamic reticular nucleus itself. To determine if neurons in the ep differed from those in the thalamic reticular nucleus in Alligator mississippiensis, the ep was investigated using Golgi methodology. The morphology and soma size of neurons in the ep differed from those in the thalamic reticular nucleus and indicated that these two areas are indeed separate neuronal aggregates. Based on these data and the known relationships of the zona incerta to surrounding structures in mammals, the ep of crocodilians is suggested to be the counterpart of the zona incerta of mammals.

Thalamic reticular nucleus in Alligator mississippiensis: Soma and dendritic morphology.

The thalamic reticular nucleus (TRN) is a critical structure influencing information transfer to the forebrain. In crocodilians, the TRN shares many features with its mammalian counterpart. One area that has not been explored is how individual neurons in the crocodilian TRN compare with those found in mammals. In mammals, TRN neurons are aligned parallel to the external border of the dorsal thalamus, have their dendrites oriented perpendicular to the fibers in the internal capsule, have fine, filamentous dendritic appendages, are either bipolar or multipolar, and are commonly considered to be a homogeneous morphological population of cells. To investigate the cellular morphology of the TRN complex, a Golgi analysis was undertaken in Alligator mississippiensis. This study examined features that have been used in mammals. In Alligator, the four TRN divisions are the dorsal peduncular nucleus, the perireticular nucleus, the interstitial nucleus, and the neurons in the medial forebrain bundle associated with the interstitial nucleus. In crocodilians, the dorsal peduncular nucleus is homologous to the TRN of mammals. From the 1787 drawn neuron profiles in the traditional three planes of section, the following were concluded. First, neurons in each part of the TRN complex in Alligator were similar in morphology. Second, each part of the TRN complex of Alligator contained a heterogenous population of cells. These variations between the cellular morphology of the dorsal peduncular nucleus of crocodilians and the TRN of mammals are speculated to partly result from differences in forebrain organization.

Magnetic resonance diffusion tensor tractography of a midbrain auditory circuit in Alligator.

Knowledge of brain circuitry is critical for understanding the organization, function, and evolution of central nervous systems. Most commonly, brain connections have been elucidated using histological and experimental methods that require animal sacrifice. On the other hand, magnetic resonance diffusion tensor imaging and associated tractography have emerged as a preferred method to noninvasively visualize brain white matter tracts. However, existing studies have primarily examined large, heavily myelinated fiber tracts. Whether tractography can visualize fiber bundles that contain thin and poorly myelinated axons is uncertain. To address this question, the midbrain auditory pathway to the thalamus was investigated in Alligator. This species was chosen because of its evolutionary importance as it is the reptilian group most closely related to birds and because its brain contains many thin and poorly myelinated tracts. Furthermore, this auditory pathway is well documented in other reptiles, including a related crocodilian. Histological observations and experimental determination of anterograde connections confirmed this path in Alligator. Tractography identified these tracts in Alligator and provided a 3-dimensional picture that accurately identified the neural elements of this circuit. In addition, tractography identified one possible unrecognized pathway. These results demonstrate that tractography can visualize circuits containing thin, poorly myelinated fibers. These findings open the door for future studies to examine these types of pathways in other vertebrates.

Thalamic Reticular Nucleus in Caiman crocodilus: Immunohistochemical Staining.

The thalamic reticular nucleus in reptiles, Caiman crocodilus, shares a number of morphological similarities with its counterpart in mammals. In view of the immunohistochemical properties of this nucleus in mammals and the more recently identified complexity of this neuronal aggregate in Caiman, this nucleus was investigated using a number of antibodies. These results were compared with findings described for other amniotes. The following antibodies gave consistent and reproducible results: polyclonal sheep anti-parvalbumin (PV), monoclonal mouse anti-PV, and polyclonal sheep anti-glutamic acid decarboxylase (GAD). In the transverse plane, this nucleus is divided into two. In each part, a compact group of cells sits on top of the fibers of the forebrain bundle with scattered cells among these fibers. In the lateral forebrain bundle, this neuronal aggregate is represented by the dorsal peduncular nucleus and the perireticular nucleus while, in the medial forebrain bundle, these parts are the interstitial nucleus and the scattered cells in this fiber tract. The results of this study are the following. First, the thalamic reticular nucleus of Caiman contains GAD(+) and PV(+) neurons, which is similar to what has been described in other amniotes. Second, the morphology and distribution of many GAD(+) and PV(+) neurons in the dorsal peduncular and perireticular nuclei are similar and suggest that these neurons colocalize these markers. Third, neurons in the interstitial nucleus and in the medial forebrain bundle are GAD(+) and PV(+). At the caudal pole of the thalamic reticular nucleus, PV immunoreactive cells predominated and avoided the central portion of this nucleus where GAD(+) cells were preferentially located. However, GAD(+) cells were sparse when compared with PV(+) cells. This immunohistochemically different area in the caudal pole is considered to be an area separate from the thalamic reticular nucleus.

Thalamic reticular nucleus in Caiman crocodilus: forebrain connections.

Forebrain connections of the thalamic reticular nucleus associated with the lateral forebrain bundle were analyzed in Caiman crocodilus. Both the compact portion, the dorsal peduncular nucleus, and the diffuse part, the perireticular region, associated with the lateral forebrain bundle, were studied. A small tracer injection into the dorsal peduncular nucleus demonstrated reciprocal connections with a restricted portion of the dorsal thalamus. Tracer placements into this nucleus retrogradely labeled cells in a caudal portion of the ventrolateral area of the telencephalon. These results are compared with similar studies in other amniotes.

Crocodilian Forebrain: Evolution and Development.

Organization and development of the forebrain in crocodilians are reviewed. In juvenile Caiman crocodilus, the following features were examined: identification and classification of dorsal thalamic nuclei and their respective connections with the telencephalon, presence of local circuit neurons in the dorsal thalamic nuclei, telencephalic projections to the dorsal thalamus, and organization of the thalamic reticular nucleus. These results document many similarities between crocodilians and other reptiles and birds. While crocodilians, as well as other sauropsids, demonstrate several features of neural circuitry in common with mammals, certain striking differences in organization of the forebrain are present. These differences are the result of evolution. To explore a basis for these differences, embryos of Alligator misissippiensis were examined to address the following. First, very early development of the brain in Alligator is similar to that of other amniotes. Second, the developmental program for individual vesicles of the brain differs between the secondary prosencephalon, diencephalon, midbrain, and hindbrain in Alligator. This is likely to be the case for other amniotes. Third, initial development of the diencephalon in Alligator is similar to that in other amniotes. In Alligator, alar and basal parts likely follow a different developmental scheme.

Dorsal thalamic nuclei in Caiman crocodilus.

In Caiman crocodilus, identification of nuclei that comprise the dorsal thalamus was determined by: injections of retrograde tracers into cortex/pallium; injections of retrograde tracers into the noncortical telencephalon; and injections of anterograde tracers into thalamic nuclei. With the exception of nucleus dorsolateralis anterior, which has bilateral projections, all other dorsal thalamic nuclei send axons to terminate in the ipsilateral telencephalon. Nuclei that only projected to cortex/pallium were: dorsolateralis anterior; diagonalis; and dorsal geniculate. Neuronal aggregates that send axons that terminated in the dorsolateral area (dorsal ventricular ridge) included: rotundus; reuniens pars centralis and pars diffusa; medialis complex posterior; posterocentralis; and area ventrolateralis. Medialis complex anterior axons ended in the ventrolateral area (basal ganglia). Nucleus dorsomedialis projected to both cortex/pallium and the dorsolateral area. Based on the locus of telencephalic termination and fiber trajectory from the dorsal thalamus, nuclei of the dorsal thalamus were divided into several groups.

Geometry of saccular cerebral aneurysms not associated with a branch vessel.

OBJECTIVE: Saccular cerebral aneurysms located at nonbranching sites are uncommon. Their distribution, morphological features, and presence of a branch vessel or a tiny perforator(s) separate from the aneurysm neck were investigated. METHODS: From a series of 303 microsurgically clipped saccular cerebral aneurysms, 40 aneurysms were identified at sites not related to a branch vessel. RESULTS: The distribution of aneurysms at nonbranching sites was internal carotid: 21 of 40 (52.5%); main stem of the middle cerebral artery/secondary branch of the middle cerebral artery: 6 of 40 (15%); anterior cerebral artery: 1 of 40 (2.5%); pericallosal artery: 1 of 40 (2.5%); pericallosal/callosal marginal: 3 of 40 (7.5%); vertebral artery: 1 of 40 (2.5%); posterior cerebral artery: 1 of 40 (2.5%); posterior cerebral artery/secondary branch of the posterior cerebral artery: 1 of 40 (2.5%); anterior inferior cerebellar artery: 1 of 40 (2.5%); and distal posterior inferior cerebellar artery: 1 of 40 (2.5%). Branch vessels were seen in 5 cases, and small perforating vessels were observed in 2 instances. CONCLUSIONS: Saccular aneurysms occurring at nonbranching sites are uncommon. Their geometry is particularly favorable for flow directed stents and is most amenable to aneurysms located on large-diameter conducting vessels such as the internal carotid, vertebral, and vertebrobasilar vessels. Smaller parent arteries harboring this type of aneurysm will require new technology to maintain patency of these more distal vessels. If endovascular techniques cannot achieve aneurysm sac obliteration, then open craniotomy and aneurysm clipping will provide a satisfactory alternative.

Perforator and secondary branch origin in relation to the neck of saccular, cerebral bifurcation aneurysms.

OBJECTIVE: Perforator and secondary branch origin in relation to the neck of cerebral, saccular bifurcation aneurysms were analyzed. These two features were considered important for treatment. METHODS: From a series of microsurgically clipped saccular cerebral aneurysms, 142 bifurcation aneurysms had detailed imaging studies and operative records that could be analyzed. RESULTS: The incidence of perforator origin from the aneurysm neck was as follows: basilar, 1/15 (7%); internal carotid artery bifurcation, 4/23 (17%); main stem of the middle cerebral artery/secondary branch of the middle cerebral artery, 6/52 (12%); anterior communicating artery region, 5/46 (11%); and distal bifurcation vessels, 0/6 (0%). Aneurysms arising from the anterior communicating artery between the anterior cerebral arteries had a high incidence of perforator origin from the aneurysm neck. The location of secondary branch origin from the aneurysm neck varied depending on the aneurysm group. CONCLUSION: Perforator origin from the aneurysm neck was infrequent. A subgroup of anterior communicating artery region aneurysms had a high incidence of perforator origin from the aneurysm neck. Although protection of these neck perforators will be difficult, their identification may be even more challenging. Secondary branch origin from the aneurysm neck varied depending on the aneurysm group. Advanced endovascular techniques are needed to obliterate aneurysms in which the secondary branch(es) arise from the aneurysm neck. If this is not possible, craniotomy and clip ligation will be required if complete aneurysm obliteration is the goal.

CHARACTERIZING CALCIUM INFLUX VIA VOLTAGE- AND LIGAND-GATED CALCIUM CHANNELS IN EMBRYONIC ALLIGATOR NEURONS IN CULTURE.

Vertebrate brains share many features in common. Early in development, both the hindbrain and diencephalon are built similarly. Only later in time do differences in morphology occur. Factors that could potentially influence such changes include certain physiological properties of neurons. As an initial step to investigate this problem, embryonic Alligator brain neurons were cultured and calcium responses were characterized. The present report is the first to document culture of Alligator brain neurons in artificial cerebrospinal fluid (ACSF) as well as in standard mammalian tissue culture medium supplemented with growth factors. Alligator brain neuron cultures were viable for at least 1 week with unipolar neurites emerging by 24 hours. Employing Fura-2 AM, robust depolarization-induced calcium influx, was observed in these neurons. Using selective blockers of the voltage-gated calcium channels, the contributions of N-, P/Q-, R-, T-, and L-type channels in these neurons were assessed and their presence documented. Lastly, Alligator brain neurons were challenged with an excitotoxic stimulus (glutamate + glycine) where delayed calcium deregulation could be prevented by a classical NMDA receptor antagonist.

Angiographically visible and invisible arteriovenous malformation in the same patient.

A pathologically confirmed angiographically visible and invisible arteriovenous malformation in the same patient is described. The potential clinical significance of these observations is detailed and discussed.