A review of mammalian carcinogenicity study design and potential effects of alternate test procedures on the safety evaluation of food ingredients.

Extensive experience in conducting long term cancer bioassays has been gained over the past 50 years of animal testing on drugs, pesticides, industrial chemicals, food additives and consumer products. Testing protocols for the conduct of carcinogenicity studies in rodents have been developed in Guidelines promulgated by regulatory agencies, including the US EPA (Environmental Protection Agency), the US FDA (Food and Drug Administration), the OECD (Organization for Economic Co-operation and Development) for the EU member states and the MAFF (Ministries of Agriculture, Forestries and Fisheries) and MHW (Ministry of Health and Welfare) in Japan. The basis of critical elements of the study design that lead to an accepted identification of the carcinogenic hazard of substances in food and beverages is the focus of this review. The approaches used by entities well-known for carcinogenicity testing and/or guideline development are discussed. Particular focus is placed on comparison of testing programs used by the US National Toxicology Program (NTP) and advocated in OECD guidelines to the testing programs of the European Ramazzini Foundation (ERF), an organization with numerous published carcinogenicity studies. This focus allows for a good comparison of differences in approaches to carcinogenicity testing and allows for a critical consideration of elements important to appropriate carcinogenicity study designs and practices. OECD protocols serve as good standard models for carcinogenicity testing protocol design. Additionally, the detailed design of any protocol should include attention to the rationale for inclusion of particular elements, including the impact of those elements on study interpretations. Appropriate interpretation of study results is dependent on rigorous evaluation of the study design and conduct, including differences from standard practices. Important considerations are differences in the strain of animal used, diet and housing practices, rigorousness of test procedures, dose selection, histopathology procedures, application of historical control data, statistical evaluations and whether statistical extrapolations are supported by, or are beyond the limits of, the data generated. Without due consideration, there can be result conflicting data interpretations and uncertainty about the relevance of a study's results to human risk. This paper discusses the critical elements of rodent (rat) carcinogenicity studies, particularly with respect to the study of food ingredients. It also highlights study practices and procedures that can detract from the appropriate evaluation of human relevance of results, indicating the importance of adherence to international consensus protocols, such as those detailed by OECD.

Superior colliculus of the tree shrew: a structural and functional subdivision into superficial and deep layers.

Superficial lesions of the superior colliculus produced deficits in form discrimination, while deeper lesions produced, in addition, an inability to track objects. These two syndromes were related to an anatomical subdivision: Superficial lesions resulted in anterograde degeneration in the visual thalamus, whereas lesions confined to the deeper layers produced degeneration in the nonvisual thalamus and in brainstem motor areas.

Excitatory and inhibitory circuitry in the superficial gray layer of the superior colliculus.

Stratum griseum superficiale (SGS) of the superior colliculus receives a dense cholinergic input from the parabigeminal nucleus. In this study, we examined in vitro the modulatory influence of acetylcholine (ACh) on the responses of SGS neurons that project to the visual thalamus in the rat. We used whole-cell patch-clamp recording to measure the responses of these projection neurons to electrical stimulation of their afferents in the stratum opticum (SO) before and during local pressure injections of ACh. These colliculothalamic projection neurons (CTNs) were identified during the in vitro experiments by prelabeling them from the thalamus with the retrograde axonal tracer wheat germ agglutinin-apo-HRP-gold. In a group of cells that included the prelabeled neurons, EPSCs evoked by SO stimulation were significantly reduced by the application of ACh, whereas IPSC amplitudes were significantly enhanced. Similar effects were observed when the nicotinic ACh receptor agonist lobeline was used. Application of the selective GABA(B) receptor antagonist 3-[[(3,4-dichlorophenyl)-methyl]amino]propyl](diethoxymethyl)phosphinic acid blocked ACh-induced reduction in the evoked response. In contrast, the ACh-induced reduction was insensitive to application of the GABA(A) receptor antagonist bicuculline. The ACh-induced reduction was also diminished by bath application of muscimol at the low concentrations that selectively activate GABA(C) receptors. Because GABA(C) receptors may be specifically expressed by GABAergic SGS interneurons (Schmidt et al., 2001), our results support the hypothesis that ACh reduces CTN activity by nicotinic receptor-mediated excitation of local GABAergic interneurons. These interneurons in turn use GABA(B) receptors to inhibit the CTNs.

Disinhibition in rat superior colliculus mediated by GABAc receptors.

The stratum griseum superficiale (SGS) of the superior colliculus contains a high concentration of the recently described GABA(C) receptor. In a previous study, it was postulated that activation of these receptors on inhibitory interneurons functions to disinhibit projection cells that relay visual information to the thalamus and brainstem. To test this model, we used in vitro whole-cell patch-clamp methods to measure effects of GABA and muscimol on EPSCs and IPSCs evoked in rat SGS by electrical optic layer stimulation. The neurons were filled with biocytin for later morphological characterization. As expected, bath applications of GABA and muscimol always strongly depressed evoked PSCs at concentrations of >100 and >1 micrometer, respectively. However, at lower agonist concentrations, which most likely activate GABA(C) but not GABA(A) receptors, effects were not uniform. Evoked responses were suppressed by both agonists in 48% of the neurons, whereas the remaining cells exhibited enhanced responses with increased evoked EPSCs, decreased evoked IPSCs, or both types of change. Most morphologically identified cells with suppressed responses (14 of 17 cells) had morphological characteristics of putative GABAergic interneurons, whereas almost all cells with enhanced responses (8 of 10 cells) had morphological characteristics of projection cells. Finally, all effects of GABA and muscimol at low concentrations were blocked by (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid, a specific GABA(C) receptor antagonist, but not by the specific GABA(A) receptor antagonist bicuculline. Taken together, these results indicate that in SGS, GABA(C) receptors are predominantly expressed by GABAergic neurons and that activation of these receptors leads to disinhibition of SGS projection cells.

Interlaminar connections of the superior colliculus in the tree shrew. I. The superficial gray layer.

One of the most persistent problems in the study of the superior colliculus is the relationship between its superficial and deep layers. The superficial tier of layers is considered to be visuosensory in function, whereas the deep tier is multisensory and has premotor functions. This fundamental distinction is the primary basis for the view that a visually triggered shift in the direction of gaze depends on the transfer of information from sensory cells in the superficial tier to premotor cells in the deep tier. The goal of the present experiments was to examine the interlaminar projections of the superficial gray layer in the tree shrew Tupaia belangeri. We used biocytin as the marker for tracing the pathways. The tree shrew was chosen because its large and distinctly laminated superior colliculus facilitates the task of examining connections between the layers. Biocytin was used because of its sensitivity and because it allowed us to place very small injections restricted entirely to the superficial gray layer. The results demonstrated that a prominent pathway originates in the superficial gray layer and terminates in stratum opticum. In comparison, the projection from the superficial gray layer to the layers beneath stratum opticum is extremely sparse. The pathway from the superficial gray layer to stratum opticum has a columnar distribution, extending about 100 microns rostrally and caudally from the center of the injection site. There were no signs of more remote intracollicular connections, nor of patches or bands of terminals. The biocytin injection sites also labeled pathways to nuclei as distant from the superior colliculus as the diencephalon, including the dorsal and ventral lateral geniculate bodies, and the pulvinar. The results suggest that stratum opticum may serve as a link between the superficial gray layer and the deeper layers.

Collateral projections of predorsal bundle cells of the superior colliculus in the rat.

The deep layers of the superior colliculus contain cells which are premotor in the sense that they respond prior to the onset of shifts in gaze and send axons, by way of a pathway called the predorsal bundle, to the contralateral brainstem gaze centers and cervical spinal cord. Previous studies have suggested that these cells also contribute to other efferent pathways which arise in the deep layers. The present study examines the contributions of the cells of origin of the predorsal bundle to these additional pathways as a step toward understanding their roles in gaze mechanisms. In one series of experiments, retrograde tracers were used to compare the laminar distribution of predorsal bundle cells with the distributions of the cells of origin of three other pathways: those that project to the intralaminar region of the dorsal thalamus, those that project to the contralateral superior colliculus, and those that project to the ipsilateral brainstem tegmentum. Predorsal bundle cells were found primarily in stratum griseum intermedium sublayer b. This distribution overlaps extensively with the distribution of colliculus cells that project to the intralaminar region of the thalamus. In contrast, the majority of the colliculus cells that project to either the contralateral superior colliculus or the ipsilateral brainstem tegmentum do not overlap extensively with the predorsal bundle cells; instead, they are primarily located dorsal or ventral to sublayer b of stratum griseum intermedium. In a second series of experiments, two regions were injected with different retrograde fluorescent traces in single animals in order to study the collateral projections of the cells of origin of these pathways. The results indicate that many predorsal bundle cells project to the intralaminar region of the dorsal thalamus but that only a few contribute to the tectotectal pathway. The results also indicate that few tectotectal cells contribute to the ipsilateral tectobulbar pathway.

The nigral projection to predorsal bundle cells in the superior colliculus of the rat.

Predorsal bundle cells give rise to the major efferent pathway from the superior colliculus to the premotor centers of the brainstem and spinal cord responsible for initiating orienting movements. The activity of predorsal bundle cells is profoundly influenced by an inhibitory pathway from substantia nigra pars reticulata that uses gamma aminobutyric acid (GABA) as a neurotransmitter. The present study examines the morphological basis for this influence of substantia nigra on predorsal bundle cells in the rat. In the first experiments, the laminar distributions of the nigrotectal tract terminals and the predorsal bundle cells were compared. The predorsal bundle cells were labeled by the retrograde axonal transport of horseradish peroxidase from either the decussation of the predorsal bundle or the cervical spinal cord, while the terminations of the pathway from substantia nigra pars reticulata were labeled by anterograde axonal transport from the substantia nigra. Either horseradish peroxidase, wheat germ agglutinin conjugated to horseradish peroxidase, or Phaseolus vulgaris leucoagglutinin were used as anterograde tracers. The results showed that the distributions of both the predorsal bundle cells and the nigrotectal terminals are restricted almost entirely to the intermediate grey layer and that they overlap extensively. Predorsal bundle cells varied in size. Within the areas of maximum overlap, the majority, regardless of size, was closely apposed by nigrotectal terminals. In a second series of experiments, the synaptic contacts between nigrotectal terminals and the tectospinal component of the predorsal bundle were examined in tissue in which both the terminals and the tectospinal cells were labeled for electron microscopy. In the final experiments, the distribution and fine structure of the nigrotectal terminals were compared to those of terminals that had been labeled immunocytochemically with an antibody to glutamic acid decarboxylase, the synthesizing enzyme for GABA. The results showed that nigrotectal terminals contain large numbers of mitochondria and pleomorphic vesicles, and form synaptic contacts with the somas and proximal dendrites of tectospinal cells. These synapses have modest postsynaptic densities. In both their distribution and fine structure, these terminations resemble the glutamic acid decarboxylase immunoreactive terminals that contact tectospinal cells. Taken together, these results support the view that the nigrotectal tract is an important source of GABAergic input to most, if not all, predorsal bundle cells.

Efferent projections of the main and the accessory olfactory bulb in the tree shrew (Tupaia glis).

The projections of the main and the accessory olfactory bulb in the tree shrew (Tupaia glis) have been analyzed with anterograde degeneration and autoradiographic methods for identifying axonal projections, and with the horseradish peroxidase method for identifying the distribution of neurons from which these projections originate. The cytoarchitectonic features of the paleocortical areas which receive projections from the main and the accessory olfactory bulb have also been described. The efferent projections of the accessory olfactory bulb are distributed to the bed nucleus of the accessory olfactory tract, the medial amygdaloid area, the posteromedial cortical amygdaloid area, and to the caudal portion of the bed nucleus of the stria terminalis. In contrast, the efferent projections of the main olfactory bulb are distributed to the anterior olfactory nucleus, the tenia tecta, the olfactory tubercle, the pyriform cortex, the anterior cortical amygdaloid area, the posterolateral cortical amygdaloid area, and to the lateral entorhinal cortex. These observations are consistent with the notion that the olfactory system can be divided into at least two major subsystems: one related to the vomeronasal organ and accessory olfactory bulb, and another related to the main olfactory organ and main olfactory bulb. The paleocortical areas receiving olfactory projections have three basic layers: a superficially positioned plexiform layer (layer I), a pyramidal cell layer (layer II), and a polymorphic cell layer (layer III). The projections of both the main and the accessory olfactory bulb terminate in the outer portion of the plexiform layer (sublamina Ia). Sublamina Ia contains the distal segments of dendrites which originate from a heterogeneous population of neurons located in layer II and, to a lesser extent, layer III. Although the efferent projections of the main and the accessory olfactory bulb are segregated, evidence for a more refined topographical organization within these projections was not obtained. However, the distribution of retrogradely labeled neurons in the main olfactory bulb, following injections of horseradish peroxidase into its various paleocortical targets, indicates that the olfactory projections to these areas may not all originate from the same population of cells.

The medial geniculate body of the tree shrew, Tupaia glis. I. Cytoarchitecture and midbrain connections.

In this study of the medial geniculate body in the tree shrew eight subdivisions are identified on the basis of differences recognized in Nissl-stained material. Experiments using the methods of anterograde and retrograde axonal transport and anterograde degeneration show that each subdivision has a unique pattern of connections with the midbrain. The ventral division of the medial geniculate body contains at least two subdivisions, the ventral nucleus and the caudomarginal nucleus. The ventral nucleus is characterized by densely-packed cells and receives topographically organized projections from the central nucleus of the inferior colliculus. The caudomarginal nucleus, on the other hand, receives its major midbrain projections from the medial nucleus in the inferior colliculus. In the dorsal division four subdivisions are distinguished. The suprageniculate nucleus contains large, loosely-packed cells and receives projections from the deep layers of the superior colliculus and from the midbrain tegmentum. The dorsal nucleus receives projections from the midbrain tegmentum. The deep dorsal and anterodorsal nuclei have neurons which resemble those in the dorsal nucleus. Both receive projections from the roof nucleus of the inferior colliculus but the deep dorsal nucleus receives an additional projection from the parabrachial tegmentum. The medial division has a rostral and a caudal subdivision. The ascending projections to the rostral nucleus are from the lateral zone in the inferior colliculus and from the spinal cord. The caudal nucleus contains cells with large somas and receives projections from most of the midbrain areas which project to the other subdivisions of the medial geniculate body.

The medial geniculate body of the tree shrew, Tupaia glis. II. Connections with the neocortex.

In this study the temporal cortex of the tree shrew was subdivided on the basis of cytoarchitectonic criteria, and the connections of each subdivision with the thalamus and midbrain were analyzed with retrograde and anterograde techniques. The results indicate that, with one exception, each subdivision of the medial geniculate body projects to a separate cortical area. The primary auditory cortex receives projections from the ventral nucleus. Surrounding the primary cortex are at least five additional cytoarchitectonically distinct areas which receive projections from the remaining medial geniculate subdivisions. The evidence suggests that there is very little overlap in the projections from each of these geniculate subdivisions. An exception is the projection of the caudal nucleus of the medial division. This subdivision apparently projects to most, if not all, of the cortical target of the medial geniculate body. Although the cortical projections of the caudal nucleus overlap those of the other medial geniculate subdivisions, the laminar distribution of its terminations in cortex is different. The caudal nucleus projects primarily to layer VI whereas the other subdivisions of the medial geniculate body project primarily to layer IV and the adjacent part of layer III. Anterograde techniques were also used to study the projections from the cortex back to the thalamus and to the midbrain. The projections to the thalamus precisely reciprocate the thalamocortical connections. The projections to the midbrain are to the same areas which the preceding study (Oliver and Hall, '78) showed give rise to ascending projections to the medial geniculate body. An exception is the central nucleus of the inferior colliculus which apparently does not receive a projection from the temporal cortex.

The organization of the pulvinar in the grey squirrel (Sciurus carolinensis). I. Cytoarchitecture and connections.

The posterior neocortex in the grey squirrel, Sciurus carolinensis, includes an extensive region which receives projections from the pulvinar. Previous studies have demonstrated that this cortical region can be subdivided on the basis of differences in cytoarchitecture and electrophysiologically defined representations of the visual field. The main purpose of the present paper was to determine whether these cortical subdivisions could be related to corresponding subdivisions in the pulvinar. The methods used to trace connections included anterograde degeneration, anterograde axonal transport of tritiated amino acids and the retrograde axonal transport of horseradish peroxidase. The results indicate that the pulvinar in this species contains at least three main subdivisions which can be distinguished by their cytoarchitecture and their patterns of connections. A caudal subdivision contains large, evenly-spaced neurons and receives bilateral input from the superficial, retinal-recipient layers of the superior colliculus. This caudal subdivision has reciprocal interconnections with a cytoarchitectonically distinct area in the temporal cortex. A rostro-lateral subdivision contains smaller, more lightly stained neurons which tend to form clusters. This subdivision receives only ipsilateral tectal input and projects to occipital area 18. This subdivision does not receive input from areas 17, 18, and 19, or from the temporal cortex. Finally, a rostro-medial subdivision is cytoarchitectonically similar to the rostro-lateral subdivision but receives little, if any, input from the superior colliculus. This rostro-medial area does, however, receive corticofugal projections from occipital areas 17, 18, and 19, and projects to area 19. These patterns of connections suggest that each of these subdivisions has close associations with the visual system. The question of whether similar subdivision are present in the visual thalamus of other species is discussed.

The organization of the pulvinar in the grey squirrel (Sciurus carolinensis). II. Synaptic organization and comparisons with the dorsal lateral geniculate nucleus.

The purpose of these experiments was to compare the synaptic organization of the subdivisions of the pulvinar defined in the preceding paper (Robson and Hall, '77) with each other and with the organization present in the dorsal lateral geniculate nucleus. The electron microscope was used to analyze normal synaptic arrangements and degenerating axonal terminals resulting from lesions. The dorsal lateral geniculate nucleus in the grey squirrel contains synaptic clusters similar to those described previously for other species. These clusters are characterized by large optic tract terminals which form multiple contacts onto large dendritic processes and other processes containing flat or pleomorphic vesicles. The geniculate lamina adjacent to the optic tract receives projections from the superior colliculus as well are from the retina. The terminals of the superior colliculus axons are small and medium sized and lie outside of the synaptic clusters. The retinal terminals are in the clusters. In the pulvinar, the rostro-medial subdivision contains synaptic clusters which resemble those in the lateral geniculate nucleus. These clusters contain large axon terminals which make multiple contacts onto large dendrites. However, these terminals are not contributed by an ascending sensory pathway but by axons from striate cortex. The rostro-lateral and caudal subdivisions of the pulvinar also contain synaptic clusters, but these clusters consist of a segment of a large dendrite which is ensheathed by medium-sized terminals. Since only a few of these medium sized terminals in any one cluster degenerate after tectal lesions, and none degenerate after cortical lesions, it is suggested that the morphological arrangement of these clusters may permit the convergence of axons from several sources, some of which are unidentified, onto the same dendritic segment.

The connections and laminar organization ofthe optic tectum in a reptile (lguana iguana).

The goals of this study were: (1) to describe the total pattern of projections from the optic tectum of Iguana iguana and Pseudemys scripta; and (2) to describe the contributions of particular lamina of the Iguana's optic tectum to this total pattern. Lesions were made in the optic tectum of the Iguana which damaged either all or only certain tectal laminae and, for comparison with the Iguana, lesions in the turtle's optic tectum were made which involved all laminae. The anterograde degeneration resulting from these lesions was stained with the Fink-Heimer ('67) method. The total pattern of projections from the optic tectum in the Iguana and the turtle is similar to that reported for representatives of other vertebrate classes. That is, the optic tectum gives rise to ipsilateral ascending projections to pretectal nuclei, to nucleus rotundus and to nucleus geniculatus lateralis pars ventralis of the diencephalon and, in addition, to a contralateral ascending pathway which courses via the supraoptic decussation to the contralateral diencephalon. Tectotectal connections and several descending pathways were also recognized in each species. The descending pathways include ipsilateral tectobulbar and tecto-isthmi pathways and a contralateral predorsal bundle. Lesions which damaged only certain tectal laminae in the Iguana revealed a laminar organization of the efferent projections. A lesion restricted to the superficial retinal-recipient layers, stratum griseum et album superficiale, resulted in degeneration in only nucleus isthmi pars magnocellularis and nucleus geniculatus lateralis pars ventralis. A lesion which involved both the retinal-recipient layers and stratum griseum centrale resulted in degeneration in only one additional structure, nucleus rotundus. A small lesion involving the deep periventricular layers as well as the superficial layers produced degeneration in the predorsal bundle and the ipsilateral tectobulbar tract as well as in the structures receiving input from the more superficial layers. These results are compared to the results of similar analyses of the superior colliculus in mammals.

Relationships between the nigrotectal pathway and the cells of origin of the predorsal bundle.

The goal of this study was to define the anatomical relationships between the terminal field of the nigrotectal pathway and the tectal neurons which project to contralateral brainstem gaze centers by way of the predorsal bundle. The distribution and morphology of the cells of origin for the predorsal bundle were determined by using a modification of the retrograde horseradish peroxidase technique which homogeneously filled their somas and dendrites. The terminal distribution of the nigrotectal tract was determined using both anterograde horseradish peroxidase and autoradiographic procedures. The results indicate that, in the grey squirrel (Sciurus carolinensis), the predorsal bundle cells are a heterogeneous population whose dendritic fields form a well-defined band confined to the inner half of stratum griseum intermediate. This inner sublamina also can be identified in Nissl and myelin stains. The same sublamina is the major target of the nigrotectal tract. The striking anatomical correspondence between the distribution of nigrotectal terminals and the cells projecting in the predorsal bundle supports a proposal, based on recent physiological investigations, that the nigrotectal tract plays an important role in the initiation of the saccade-related activity of the deep tectal cells (Chevalier et al., '81; Hikosaka and Wurtz, '83a-d).

The normal organization of the lateral posterior nucleus of the golden hamster.

As a first step in analyzing the influence of various afferent projections on the development of the hamster lateral posterior nucleus, its normal organization was studied using both light and electron microscopic techniques. Rostrolateral, rostromedial, and caudal subdivisions were identified. The rostrolateral subdivision receives dense projections from the ipsilateral superior colliculus and posterior neocortex, as well as sparser, more restricted projections from the contralateral colliculus and retina. The ipsilateral colliculus is by far the major source of medium-sized (M)terminals with round vesicles. These terminals synapse around the shafts of large central dendrites to form distinctive synaptic clusters. The contralateral colliculus and retina contribute a few M-terminals to the clusters. In contrast, axons from the posterior neocortex form very large (RL-)terminals with round vesicles from the posterior neocortex form very large (RL)terminals with round vesicles which synapse onto numerous appendages of single proximal dendrite, are surrounded by glial lamellae, and rarely participate in the clusters. Axons from all four sources also form small (RS)terminals with round vesicles which synapse on the shafts of small dendrites. Finally, F-terminals with flat or pleomorphic vesicles form symmetric synaptic contacts both within and outside the clusters. The only identified projection to the rostromedial subdivision is from the ipsilateral posterior neocortex, which contributes RL- and RS-terminals. F-terminals are also found, but neither M-terminals nor synaptic clusters are present. The caudal subdivision also receives RL- and RS-terminals from the ipsilateral posterior neocortex. Small inputs from the ipsilateral and contralateral colliculi are present, but their axons form only RS-terminals. No M-terminals or synaptic clusters are found. These results indicate that a large neonatal superior colliculus lesion would eliminate the vast majority of the M-terminals in the synaptic clusters of the ipsilateral lateral posterior nucleus. In subsequent studies (Crain and Hall, '80 a,b,c), we will examine how the remaining inputs from the retina, contralateral superior colliculus, and posterior neocortex contribute to the synaptic organization when it develops after such a lesion.

The organization of the lateral posterior nucleus in neonatal golden hamsters.

The present series of experiments was designeneonatal golden hamsters.

The organization of afferents to the lateral posterior nucleus in the golden hamster after different combinations of neonatal lesions.

After a neonatal lesion of the ipsilateral superior colliculus, the projections to the lateral posterior nucleus from the contralateral superior colliculus and retina expand their terminal fields until they share a common border. In the first experiment described in this paper, we removed both superior colliculi at birth and used the Fink-Heimer method to show that the optic tract projection could expand even further and enter the region which would have been occupied by the terminals of the crossed colliculus projection. Similarly, in the second experiment, we showed that the crossed collicular projection could be increased even more if the contralateral eye as well as the ipsilateral colliculus was removed at birth. Another result of a neonatal superior colliculus lesion is that the projection from the optic tract shares a border with the posterior neocortical projection. In the third experiment, we removed both the ipsilateral superior colliculus and the posterior neocortex at birth and demonstrated that the optic tract projection expanded more than after an ipsilateral colliculus lesion alone. Our results support the hypotheses that the projections from the ipsilateral and contralateral superior colliculi and the retina compete for synaptic space in the lateral posterior nucleus, and that a similar competition between the retinal and cortical projections may also occur.

The organization of the lateral posterior nucleus of the golden hamster after neonatal superior colliculus lesions.

The reorganization of the adult hamster's lateral posterior nucleus after neonatal superior colliculus lesions was studied using primarily light and electron microscopic degeneration techniques. Two types of experiments were conducted. First, the distributions of the remaining afferents from the contralateral superior colliculus, the cotralateral retina, and the ipsilateral posterior neocortex were determined using the Fink-Heimer ('67) technique. Normally the projections from the contralateral superior colliculus and retina are sparse and restricted to small areas in the rostrolateral subdivision. After neonatal lesions of the ipsilateral colliculus, however, these two minor projections greatly increase in density and expand to share a common border. In contrast, the normal projection from the posterior neocortex is dense throughout the rostrolateral subdivision. After a neonatal colliculus lesion, however, this projection is greatly decreased in the region occupied by the optic tract terminals. Second, the ultrastructural organizatin of the rostrolateral subdivision was studied in adult animals whhich had received neonatal colliculus lesions. Normally, this region is characterized by synaptic clusters in which numerous medium-sized terminals (M-terminals), almost all from the ipsilateral colliculus, synapse around the shaft of a large central dendrite. The contralateral colliculus and retina normally contribute only a few M-terminals. After a neonatal colliculus lesion, typical clusters still form, but now the expanded projections from the contralateral colliculus and retina contribute numerous M-terminals. The cortex does not contribute M-terminals in either normal or experimental animals. These results suggest that the afferents to the rostrolateral subdivision normally compete for synaptic space. The various factors that might be involved in determining the outcome of such competition are discussed.

The organization of central auditory pathways in a reptile, Iguana iguana.

The present experiments were designed to trace the central auditory pathways in an extant reptile, the New Worlkd lizard--Iguana iguana, utilizing anterograde axonal degeneration stained by the Fink-Heimer ('67) method and the retrograde axonal transport of horseradish peroxidase (LaVail and LaVail, '74). Beginning with the projections of the auditory portion of the VIIIth nerve, the ascending pathways were traced through successive relay nuclei to the telencephalon. The auditory portion of the VIIIth nerve projects to two nuclei in the dorsomedial medulla-nucleus angularis and nucleus magnocellularis medialis. These two nuclei together with a third cll group, nucleus magnocellularis lateralis (intercalated between nucleus angularis and nucleus magnocellularis medialis), have been referred to as the auditory tubercle in previous studies (cf. Miller, '75). The axonal degeneration following large lesions of the auditory tubercle and small lesions of nucleus angularis demonstrated the second order auditory pathways. Fibers leave nucleus angularis ventrally and travel to the ventral surface of the medulla where they cross the midline and ascend to the midbrain in pathways resembling the trapezoid body and the lateral lemniscus of mammals. Along these pathways, terminal arborizations of some fibers were seen in three lower brainstem nuclei while other fibers ascent to the midbrain and terminate in the central nucleus of the torus semicircularis. Experiments in which horseradish peroxidase injections were made in the torus semicircularis demonstrated that nucleus angularis is a primary source of second order auditory fibers to the midbrain and, in addition, that two of the lower brainstem targets of the auditory tubercle project to the torus semicircularis. These lower brainstem pathways were shown to be associated with the auditory system by electrophysiologically recording sound-evoked responses from clusters of cells in the torus semicircularis. Ascending fibers arising from the central nucleus of the torus semicircularis were followed rostrally where they entered the dorsal thalamus and terminated throughout nucleus medialis. Finally, a thalamotelencephalic auditory pathway was traced from nucleus medialis into the lateral forebrain bundle. Terminations of this pathway from nucleus medialis were seen in the medial dorsal ventricular ridge and in the striatum. It was concluded that the ascending auditory pathways of the iguana bear a remarkable resemblance to both the mammalian and avian auditory pathways from the level of the first order neurons in the VIIIth nerve to the level of the telencephalon. At the same time, there are important specializations of the auditory system in birds and mammals such as the development of particular lower brainstem nuclei. Nevertheless, a basic plan for the organization of the auditory system in terrestrial vertebrates can be recognized which invites comparisons with the vertebrate classes that remained in aquatic habitats...

Subcortical connections of the superior colliculus in the mustache bat, Pteronotus parnellii.

The mustache bat, Pteronotus parnellii, depends on echolocation to navigate and capture prey. This adaptation is reflected in the large size and elaboration of brainstem auditory structures and in the minimal development of visual structures. The superior colliculus, usually associated with orienting the eyes, is nevertheless large and well developed in Pteronotus. This observation raises the question of whether the superior colliculus in the echolocating bat has evolved to play a major role in auditory rather than visual orientation. The connections of the superior colliculus in Pteronotus were studied with the aid of anterograde and retrograde transport of wheat germ agglutinin conjugated to HRP. These results indicate that the superior colliculus of Pteronotus is composed almost entirely of the layers beneath stratum opticum. The retinal projection is restricted to a very thin zone just beneath the pial surface. Prominent afferent pathways originate in motor structures, particularly the substantia nigra and the deep nuclei of the cerebellum. Sensory input from the auditory system originates in three brainstem nuclei: the inferior colliculus, the anterolateral periolivary nucleus, and the dorsal nuclei of the lateral lemniscus. The projections from these auditory structures terminate mainly in the central tier of the deep layer. The most prominent efferent pathways are those to medial motor structures of the contralateral brainstem via the predorsal bundle and to the ipsilateral midbrain and pontine tegmentum via the lateral efferent bundle. Ascending projections to the diencephalon are mainly to the medial dorsal nucleus and zona incerta. Thus, the superior colliculus in Pteronotus possesses well-developed anatomical connections that could mediate reflexes for orienting its ears, head, or body toward objects detected by echolocation.