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The Combat Ready Clamp is indicated to stop difficult inguinal bleeding on the battlefield, the most common type of junctional bleeding and now the most common cause of preventable battlefield death. The purpose of the present study is to report the data of clamp development to help appliers use it correctly.Wake Forest University investigators used a cadaver model to test the clamp's ability to control hemorrhage. Ten fresh cadavers were made to simulate inguinal and popliteal wound bleeding. Blood simulant was pumped to quantify device effectiveness in testing. Points of application included proximal pressure point control of popliteal, inguinal, and bilateral bleeding.Clamp use promptly controlled pulsing arterial hemorrhages from inguinal, popliteal, and bilateral wounds. The device, when placed on the common iliac artery, stopped all ipsilateral distal bleeding.The evidence of how the clamp works in the cadaver model showed that clamp use can plausibly be tailored to control inguinal hemorrhage from one wound, control two ipsilateral wounds with hemorrhage from one artery (e.g., common iliac artery), and control bilateral inguinal wounds (compression of the origins of bilateral common iliac arteries).

Calretinin (CR) is a calcium-binding protein that plays an important role in the homeostasis of intracellular calcium concentration in the auditory pathway. To test if hearing loss could lead indirectly to modifications in levels of this calcium-binding protein in neurons and neuropilar structures outside of the lemniscal auditory pathway, CR-immunostaining was evaluated in the superior colliculus (SC) in adult ferrets at 1, 20 and 90 days after unilateral cochlear ablation. The results demonstrate that within 24 h there was a significant increase in CR-immunostaining in ablated animals as indicated by an increase in the mean gray level of immunostaining in the deep, multisensory layers of the contralateral SC compared to the ipsilateral side and control ferrets. This upregulation was evident in both neurons and neuropil and did not change at the two subsequent time points. In contrast, there was no change in the superficial layers of the SC which have visual properties but no auditory inputs. These findings suggest that upregulation of CR levels within neurons and neuropil in the contralateral deep SC is subject to modifications by activity in multisynaptic auditory pathways. Therefore, cochlear-driven activity appears to affect calcium-binding protein levels not only in auditory nuclei but also in other neural structures whose response properties may be influenced by auditory-related activity.

Axonal projections from the dorsal nucleus of the lateral lemniscus (DNLL) distribute contralaterally in a pattern of banded layers in the central nucleus of the inferior colliculus (IC). The banded pattern of DNLL projections is already in the IC by onset of hearing in postnatal rat pups. Previously, it was shown that unilateral cochlear ablation in neonatal rat pups disrupted the banded pattern in IC for the projections of the DNLL contralateral to the ablation but not those of the DNLL ipsilateral to the ablation. In the present study, bilateral cochlear ablation or sham surgery was performed at postnatal day 9 (P9) after which rat pups were killed at P12 and the brains removed to study axonal projections of the DNLL. A lipophilic carbocyanine dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), was placed in the dorsal tegmental commissure of Probst to label decussating DNLL axons that end in the central nucleus of the contralateral IC. The distribution of labeled fibers across the central nucleus of the IC was analyzed in digital images by comparing the pattern of labeling with a sine model of periodic distribution of banded layers. In the control group, labeled axons formed a regular pattern of dense banded layers in IC. In the bilateral cochlear ablation group, labeled axons in the IC were distributed diffusely and there was little or no regular pattern of dense bands of axonal labeling. The influence of the cochlea on developing auditory circuits possibly mediated by activity-dependent mechanisms is discussed.

The central nucleus of the inferior colliculus (IC) is a laminated structure that receives multiple converging afferent projections. These projections terminate in a layered arrangement and are aligned with dendritic arbors of the predominant disc-shaped neurons, forming fibrodendritic laminae. Within this structural framework, inputs terminate in a precise manner, establishing a mosaic of partially overlapping domains that likely define functional compartments. Although several of these patterned inputs have been described in the adult, relatively little is known about their organization prior to hearing onset. The present study used the lipophilic carbocyanine dyes DiI and DiD to examine the ipsilateral and contralateral projections from the lateral superior olivary (LSO) nucleus to the IC in a developmental series of paraformaldehyde-fixed kitten tissue. By birth, the crossed and uncrossed projections had reached the IC and were distributed across the frequency axis of the central nucleus. At this earliest postnatal stage, projections already exhibited a characteristic banded arrangement similar to that described in the adult. The heaviest terminal fields of the two inputs were always complementary in nature, with the ipsilateral input appearing slightly denser. This early arrangement of interdigitating ipsilateral and contralateral LSO axonal bands that occupy adjacent sublayers supports the idea that the initial establishment of this highly organized mosaic of inputs that defines distinct synaptic domains within the IC occurs largely in the absence of auditory experience. Potential developmental mechanisms that may shape these highly ordered inputs prior to hearing onset are discussed.

During postnatal development, ascending and descending auditory inputs converge to form fibrodendritic layers within the central nucleus of the inferior colliculus (IC). Before the onset of hearing, specific combinations of inputs segregate into bands separated by interband spaces. These bands may define functional zones within the IC. Previous studies in our laboratory have shown that unilateral or bilateral cochlear ablation at postnatal day 2 (P2) disrupts the development of afferent bands from the dorsal nucleus of the lateral lemniscus (DNLL) to the IC. These results suggest that spontaneous activity propagated from the cochlea is required for the segregation of afferent bands within the developing IC. To test if spontaneous activity from the cochlea also may be required to maintain segregated bands of DNLL input, we performed cochlear ablations in rat pups at P9, after DNLL bands already are established. All animals were killed at P12 and glass pins coated with carbocyanine dye, DiI (1,1′-dioctodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate), subsequently were placed in the commissure of Probst to label the crossed projections from both DNLLs. When compared with surgical controls, experimental results showed a similar pattern of DNLL bands in the IC contralateral to the ablated cochlea, but a disruption of DNLL bands in the IC ipsilateral to the cochlear ablation. The present results suggest that cochlear ablation after DNLL bands have formed may affect the maintenance of banded DNLL projections within the central nucleus of the IC.

Although intrinsic organization in the inferior colliculus (IC) has been surveyed in a variety of species, current knowledge of synaptogenesis within the mammalian inferior colliculus is limited. The present study surveyed the ultrastructure of the central nucleus of the inferior colliculus in postnatal day (P) P4, P7, P14, and P28 ferrets, prior to the onset of hearing at the end of the first postnatal month with the goal of beginning to characterize the time course of synapse formation in relation to the development of afferent projection patterns within the IC. Results suggest that initial synaptogenesis has occurred in the IC by P4 and continues during the period when maturation of the distribution of axons from brainstem auditory nuclei is taking place.

Neurons of the cochlear nuclei receive axosomatic endings from primary afferent fibers from the cochlea and have projections that diverge to form parallel ascending auditory pathways. These cells are characterized by neurochemical phenotypes such as levels of calretinin. To test whether or not early deafferentation results in changes in calretinin immunostaining in the cochlear nucleus, unilateral cochlear ablations were performed in ferrets soon after hearing onset (postnatal day [P]30–P40). Two months later, changes in calretinin immunostaining as well as cell size, volume, and synaptophysin immunostaining were assessed in the anteroventral (AVCN), posteroventral (PVCN), and dorsal cochlear nucleus (DCN). A decrease in calretinin immunostaining was evident ipsilaterally within the AVCN and PVCN but not in the DCN. Further analysis revealed a decrease both in the calretinin-immunostained neuropil and in the calretinin-immunostained area within AVCN and PVCN neurons. These declines were accompanied by significant ipsilateral decreases in volume as well as neuron area in the AVCN and PVCN compared with the contralateral cochlear nucleus and unoperated animals, but not compared with the DCN. In addition, there was a significant contralateral increase in calretinin-immunostained area within AVCN and PVCN neurons compared with control animals. Finally, a decrease in area of synaptophysin immunostaining in both the ipsilateral AVCN and PVCN without changes in the number of boutons was found. The present data demonstrate that unilateral cochlear ablation leads to 1) decreased immunostaining of the neuropil in the AVCN and PVCN ipsilaterally, 2) decreased calretinin immunostaining within AVCN and PVCN neurons ipsilaterally, 3) synaptogenesis in the AVCN and PVCN ipsilaterally, and 4) increased calretinin immunostaining within AVCN and PVCN neurons contralaterally.

In this study, we used image analysis to assess changes in calretinin immunoreactivity in the lateral (LSO) and medial (MSO) superior olivary nuclei in ferrets 2 months after unilateral cochlear ablations at 30–40 days of age, soon after hearing onset. These two nuclei are the first significant sites of binaural convergence in the ascending auditory system, and both receive direct projections from the deafferented cochlear nucleus. Cochlear ablation results in a decrease in the overall level of calretinin immunostaining within the LSO ipsilaterally compared with the contralateral side and with control animals and within the MSO bilaterally compared with control ferrets. In addition, the level of calretinin immunostaining ipsilaterally within neurons in the LSO was significantly less in cochlear ablated than control animals. In contrast, there was no effect of cochlear ablation on the level of calretinin immunostaining within neurons either in the contralateral LSO or in the MSO. These results are consistent with a downregulation in calretinin within the neuropil of MSO bilaterally and LSO ipsilaterally, as well as a downregulation in calretinin within somata in the ipsilateral LSO as a result of unilateral cochlear ablation soon after hearing onset. Thus, cochlear-driven activity appears to affect calcium binding protein levels in both neuropil and neurons within the superior olivary complex. J. Comp. Neurol. 470:63–79, 2004. © 2004 Wiley-Liss, Inc.

In many systems, including ascending auditory pathways, calcium-binding proteins are markers of specific neuronal circuits. Previous studies suggest that calretinin immunostaining may be a specific marker for circuits in the inferior colliculus (IC) that code timing information. We undertook experiments to determine the changes in calretinin immunostaining in the IC that take place in response to cochlear ablation. Cochlear ablation was performed unilaterally in ferrets just after hearing onset. Animals survived for 2–3 months after ablation and brains were then processed for calretinin immunocytochemistry. The mean optical density and stained area of the calretinin immunopositive plexus in the IC were determined for five coronal sections through the right and left IC. In controls (n = 3), measurements of these parameters in the central nucleus of the IC showed symmetry between the two sides. In experimental animals (n = 8) the calretinin immunopositive plexus contralateral to the cochlear ablation was denser and larger than that in either the ipsilateral IC or in the IC of control animals. The calretinin plexus in the ipsilateral IC was slightly less dense and smaller than in controls but the differences did not reach statistical significance. IC volume measurements and synaptophysin immunostaining analysis in the central nucleus of the IC revealed no statistical differences between ablated and control animals or between the two sides in ablated animals. The significant increase in both mean optical density and immunostained area of the calretinin plexus in the IC contralateral to the cochlear ablation may reflect an upregulation in calretinin expression in the nuclei that contribute to this plexus. J. Comp. Neurol. 460:585–596, 2003. © 2003 Wiley-Liss, Inc.

In the central nucleus of the inferior colliculus (IC), afferent projections are aligned with dendritic arbors of disk-shaped cells, forming fibrodendritic layers. One feature that may serve as a guide for study of the intrinsic organization of the IC layers is the segregation of certain inputs to bands and patches within the layers of the central nucleus. In this study, we used Phaseolus leucoagglutinin as an anterograde tracer to examine the projections from the dorsal nucleus of the lateral lemniscus to the contralateral IC in adult ferrets. The labeled afferent projections distributed along the IC layers in a series of bands where there were dense endings and interband spaces where there were few if any endings. Branches of individual labeled axons that were reconstructed distributed within a single afferent band. Measurements of both the terminal density distribution and the optical density across the band were similar indicating that afferent bands were approximately 85 microm thick. Quantitative measurements of the labeled afferent bands will enhance comparison with other afferent projections and analysis of afferent development and plasticity.

One organizing principle of the auditory system is the progressive representation of best tuning frequency. Superimposed on this tonotopy are nucleotopic organizations, some of which are related to the processing of different spatial cues. In the present study, we correlated asymmetries in the outputs of the dorsal nucleus of the lateral lemniscus (DNLL) to the two inferior colliculi (ICs), with asymmetries in the inputs to DNLL from the two lateral superior olives (LSOs). The positions of DNLL neurons with crossed and uncrossed projections were plotted from cases with unilateral injections of retrograde tracers in the IC. We found an orderly dorsal-to-ventral progression to the output that recapitulated the tonotopy of DNLL. In addition, we found a nucleotopic organization in the ventral (high-frequency) part of DNLL. Neurons with projections to the ventromedial (high-frequency) part of the contralateral IC were preferentially located ventrolaterally in DNLL; those with projections to the ventromedial part of the ipsilateral IC were preferentially located ventromedially in DNLL. This partial segregation of outputs corresponded with a partial segregation of inputs from the two LSOs in cases which received closely matched bilateral injections of anterograde tracers in LSO. The ventral part of DNLL received a heavy projection medially from the opposite LSO and a heavy projection laterally from the ipsilateral LSO. The findings suggest a direct relationship in the ventral part of the DNLL between inputs from the two LSOs and outputs to the two ICs. Possible roles for this segregation of pathways in DNLL are discussed in relation to the processing of static and dynamic spatial cues.

Chemical characteristics of developing neurons in the superior olivary complex of the ferret were analyzed using immunohistochemical methods. The present report of calcium-binding proteins in the developing and adult superior olivary complex shows distinct distribution patterns for parvalbumin, calbindin, and calretinin in the lateral superior olivary nucleus (LSO) of the developing ferret that correspond to distribution patterns for different projection cell types and neurotransmitters. In the neonate, there was an initial complementary distribution of calcium-binding proteins between the shell and core of the body of the developing LSO. Parvalbumin and calbindin-immunoreactive cells were present in the shell, whereas calretinin-immunoreactive cells were restricted to the core of the LSO. Gamma amino butyric acid (GABA), but not glycine, immunoreactive cells were distributed similarly in the shell of the LSO in the neonate. There were, in addition, reciprocal medial-to-lateral gradients of parvalbumin and calbindin-immunoreactive cells in the LSO shell of the neonate. These complementary patterns in the LSO were transient, however, and by the end of the second postnatal week, each calcium-binding protein differed markedly in its cellular distribution in the superior olive, including the LSO. GABA-immunoreactive cells also were restricted transiently to the shell of the LSO in neonates. The radial segregation of transient calcium-binding expression in LSO cells was orthogonal to the medial-to-lateral axis in the LSO and, therefore, parallels fibrodendritic layers and presumed isofrequency planes of the LSO. The early postnatal segregation of calcium-binding proteins in the isofrequency axis was congruent with the gradients of contralateral and ipsilateral projection cell types in adult LSO. It seems likely that developmental mechanisms regulate expression of calcium-binding protein and neurotransmitter phenotypes and that these mechanisms operate in development within the isofrequency axis as well as along the tonotopic axis of this auditory nucleus.

The distribution and morphology of axons projecting from the medial superior olivary nucleus to the dorsal nucleus of the lateral lemniscus were studied in the adult cat. Injections of Phaseolus vulgaris-leucoagglutinin, biocytin, or dextran-rhodamine in the medial superior olivary nucleus labeled axons that ascended in the lateral lemniscus. Before entering the inferior colliculus, collateral branches of these labeled axons ended in the dorsal nucleus of the lateral lemniscus in thin, horizontal bands forming laminae that extended throughout the rostral-caudal length of the dorsal nucleus of the lateral lemniscus. A dorsal-ventral topography was apparent in the position of the lamina with respect to the injection site, but no relation between the rostral-caudal location of labeled endings and the injection site was observed. There was a divergent pattern of connections within the horizontal laminae rather than a point-to-point organization. The terminal branches of the collateral axons exhibited round or oval boutons en passant and terminaux. Individual arbors reconstructed from serial sections distributed varicosities in circumscribed domains that were only a subcomponent of the area of the afferent laminae in which they were distributed. The spatial relationships of axonal domains of several axons labeled from a single injection in the medial superior olivary nucleus suggest a mosaic pattern in the laminar connections with the dorsal nucleus of the lateral lemniscus.

Junctional body regions are too proximal for a regular limb tourniquet to fit and include the groin and axillary areas [1,2]. Recently, we described a technique of the use of the Combat Ready Clamp (Combat Medical Systems, Fayetteville, NC) in prehospital hemorrhage control and a cadaver model used in its regulatory approval, but such uses were only in the groin [3-5]. Uncontrolled upper extremity hemorrhage in war is common, disabling, and lethal; recently, we counted 833 US military casualties hospitalized with a junctional wound coded during the current wars. Of the 151 casualties with an upper extremity injury amenable to a Combat Ready Clamp in axillary use, 16.6% died of wounds (25/151; Kragh et al, unpublished data). To show users how to apply a Combat Ready Clamp to stop axillary bleeding, we illustrate its technique of use. To help users understand the device's efficacy, we report data used in its regulatory application. To test efficacy, we used Wake Forest University Medical Center's cadaver hemorrhage control model; but instead of groin bleeding, we modeled axillary bleeding in a protocol approved by the Wake Forest institutional review board [4]. After the donor bequeathed the body under ethical oversight of the School of Medicine, a male cadaver was used (age, 75 years; weight, 150 lb). The body was refrigerated until used on May 18, 2012. The right subclavian artery was cannulated with pump tubing. Water simulated blood flow at 500 mL/min at 56 beats/min (Watson-Marlow pump model 603S issue 1; Bacon Technical Industries, Inc, Concord, MA). The axillary artery was isolated in the axilla, transected, and clamped with a hemostat in between iterations of Clamp use. The artery lumen bleeding was seen before, during, and after device use. Users target the underlying axillary artery in the deltopectoral groove (where soldiers nestle rifle butts when shooting) of the abducted arm (Fig. 1).

The lateral superior olive (LSO) in ferret contains two distinct populations of principal cells, one population projecting to the ipsilateral and the other projecting to the contralateral inferior colliculus. In addition, these populations have been shown to be distinct from each other on the basis of tonotopic and isofrequency distribution within LSO, of dendritic morphology, and of neurotransmitter within the somata. The present study compared the two populations on the basis of the type of synaptic input. Laterality of projection was established using horseradish peroxidase histochemistry. Synaptic terminals contacting LSO somata identified as projecting ipsilaterally or contralaterally were quantified as round (R) or nonround (NR), representing presumptive excitatory and inhibitory input, respectively. Results indicate that the vast majority of somatic terminals contacting both projection populations are NR and, furthermore, that R terminals are significantly more likely to contact ipsilaterally than contralaterally projecting LSO cells. There is no significant difference in number of NR terminals or total number of terminals between ipsilaterally and contralaterally projecting LSO cells. These findings provide additional support to the notion that the LSO is comprised of two distinct cell populations. Moreover, they indicate a difference in the balance of somatic inhibition and excitation which may have an impact on the nature of the response properties of the two populations.

In medical curricula, difficult topics are often presented to students with varied backgrounds and therefore at different levels of preparation to learn and assimilate the information.A method is described to create an interactive primer for the study of a basic neuroscience topic to improve student preparation for and understanding of the autonomic nervous system.Action buttons or links within a PowerPoint show were used to create an interactive show. Branch points were imposed, as students answered questions correctly or incorrectly. In this way, the learning path was individualized depending on the level of comprehension.Response to the interactive learning objects has been favorably received by the students with very favorable scores on the end-of-course evaluation. Preliminary analysis of the benefit of the interactive primer on summative examination suggests modest improvement on more difficult questions.Use of the primer on the autonomic nervous system is discussed in the context of reports on the effectiveness of other blended learning and interactive learning experiences. The interactive tool described requires little facility with or support for web-based programming by faculty, and can be streamlined by creation of a PowerPoint template with pre-set interaction buttons.

Introduction There are few topics in anatomy more perplexing to first-year medical students than the autonomic nervous system. Given its complexity, this topic is amenable to approaches that may enhance student learning. In contrast to the web-based tools and technical support required with e-learning, the ability to create and deliver interactive tools with a common presentation software package can make innovative learning delivery methods, amenable to a broader base of instructors. We created an interactive learning PowerPoint tutorial for students to review prior to an anatomy lecture session. Methods As preparation for the first lecture on this topic, students may review this 25-minute PowerPoint tutorial. Be reviewing the basic objectives for the upcoming lecture, students arrive at the lecture with questions about what content is unclear and are prepared for more in-depth discussion. As a preliminary step in formally evaluating effectiveness of the interactive tutorial for student comprehension of the autonomic nervous system, results from two consecutive medical classes on a bank of standardized questions used as a part of the summative exam were compared retrospectively. Results When comparing the first round of standardized questions, the class given the interactive tutorial scored slightly higher on discriminative questions than the class with only the lecture presentation. There was no difference in performance for the two groups on competency questions. Further, instructors noticed a greater ease in communicating complex concepts of the autonomic nervous system when students had already begun to think about the anatomy and function of the system through the use of the interactive tutorial. Student feedback about this resource has been informal but the responses have been positive. Discussion The combination of an interactive learning tutorial with a lecture content in a blended learning approach has been favorably received by the students. The interactive format is engaging, complements traditional textbook reading assignments, and allows the lecturer to emphasize more difficult concepts while building on fundamental vocabulary and general principles, thereby decompressing the lecture hour. Furthermore, once the learning object is created it can be applied across a variety of instructional settings. For instance, learning objects created for the undergraduate medical education curriculum can be easily transferred for use in continuing medical education without additional preparation time and made accessible for asynchronous learning. Positive experiences with a familiar presentation format may stimulate creative learning approaches that subsequently propel faculty to explore more advanced technologies.

When ferrets are born, four weeks before the onset of hearing, few synapses are evident in the medial superior olive (MSO). The synapses present are immature and almost exclusively found in the neuropil. The MSO somata are virtually devoid of synaptic contacts but are contacted by fine glial processes that increasingly ensheathe the somata during the first postnatal week. By P12, somatic synaptogenesis in the MSO is evident. Initially the terminals contain vesicles of irregular shape, size, and distribution. The glial lamellae appear to withdraw as the synaptic contacts form but continue to cover the asynaptic portions of the cell surface. The lamellae frequently extend from an sheathing the soma to encapsulate the immature terminals. During the next two weeks, synaptic density and terminal encapsulation proceed until the somata is surrounded by encapsulated synaptic terminals as in the adult ferret MSO. While most immature terminals contain round vesicles, during the first postnatal week some terminals with nonround vesicles can be distinguished. The first distinction between types of nonround vesicle-containing terminals, i.e., pleiomorphic and ovoid, is in the second postnatal week. This distinction becomes increasingly clear and by the end of the first postnatal month, terminal types can be reliably categorized. These observations indicate that: (1) synapses are present in the MSO neuropil one month prior to the onset of hearing, (2) the major period of synaptogenesis begins approximately two weeks prior to the onset of hearing, and (3) glial lamellae ensheathe MSO somata prior to the onset of somatic synaptogenesis, withdraw as synapses form, and subsequently re-extend to encapsulate newly formed synapses.

Hippocampal (HC) function and morphology have been implicated in the pathophysiology of depression. Reduced HC volume has been observed in depressed humans, although the effect is not always significant. Studies of functional differentiation of the HC have revealed that the anterior portion is associated with emotional and anxiety-related functioning, and the posterior portion with memory processing. As such, measuring whole HC volume may mask differences seen only in the anterior or posterior HC. We used unbiased stereology to measure whole, anterior, and posterior HC volumes in 12 adult female cynomolgus macaques, half of which exhibited spontaneously occurring depressive behavior defined as a slumped/collapsed body posture with open eyes, and a relative lack of responsivity to environmental stimuli. The two groups were otherwise matched on circulating estradiol, progesterone, and cortisol levels, social status, estimated age, and body weight. Frozen postmortem HC tissue from depressed and nondepressed monkeys was serially sectioned and thionin-stained. According to established neuroanatomical guidelines and with the aid of Neurolucida software (MBF Bioscience), every tenth section throughout the extent of the HC was manually traced and used to reconstruct the 3-D models used to determine volumes. Anterior and posterior HC were delineated by the presence or absence of the uncus. No significant differences were found between depressed and nondepressed monkeys for whole or posterior HC volume, although the average HC volume was 4% smaller in depressed than nondepressed monkeys. Anterior HC volumes were significantly smaller (15.4%) in depressed compared to nondepressed monkeys. These results indicate that reduced volume in the anterior HC, an area previously implicated in emotional functioning, may be associated with a depressive phenotype in female cynomolgus macaques.

The dendritic and axonal morphology of neurons in the inferior colliculus of the cat was investigated after intracellular injection of HRP, in vivo. All injected axons gave off local collaterals, and most showed a widespread distribution and lacked a specific orientation. In contrast, the dendrites of injected neurons were distinguished by their degree of orientation and the direction of the longest axis of orientation. Dendrites showed a high, moderate, or low degree of orientation. Most highly oriented cells had their longest axis in the rostrocaudal direction with fewer in the mediolateral direction. In the central nucleus, only the rostrocaudally oriented cells correspond to the disc-shaped cells identified in Golgi preparations. Unlike most cells in our sample, the two cells that were disc-shaped had axons that were parallel to the orientation of the dendritic tree. In the dorsal cortex, rostrocaudally oriented cells also were found, but they had unoriented axons. In both the central nucleus and dorsal cortex, cells with a mediolateral axis of orientation or no specific orientation correspond to stellate cells and had axons with widespread local collaterals. These results suggest that an extensive network of local axon collaterals may contribute to neural processing within the inferior colliculus. In the central nucleus, local axons may establish connections within or across the fibrodendritic laminae. In the dorsal cortex, the local and afferent axons may form a complex reticular network. Finally, some injected cells had axons terminating locally and also entering the brachium of the inferior colliculus. This suggests that cells in the inferior colliculus may function as both interneurons and projection neurons.

The ultrastructure of the medial superior olive (MSO) was studied in the adult ferret. The synaptic terminals were categorized on the basis of morphology and their distribution determined. There are three types of synaptic terminals: R terminals, containing round vesicles; Ov terminals, containing ovoid vesicles; and P terminals, containing vesicles of varying morphologies. R terminals are the dominant terminal type on both the somata and dendrites. Ov and P terminals are equally prevalent on the dendrites; however, P terminals are significantly more common than Ov terminals on the somata. Furthermore, P terminals are significantly more common on the somata than on the dendrites. These results suggest that there is some segregation of types of afferent synapses on MSO cells.

Afferent activity modulates synaptic plasticity as well as the levels of activity-dependent molecules such as growth factors. Disruption of this activity due to deafferentation has been shown to result in an altered trophic support and consequently in changes in neuronal excitability and synaptic transmission. In the present study, to test whether lack of cochlear integrity results in changes in insulin-growth factor-1 (IGF-1) and synaptophysin immunostaining in the cochlear nucleus, the first relay structure in the auditory pathway, unilateral cochlear ablations were performed in adult ferrets. Changes in IGF-1 and synaptophysin immunostaining were assessed in the anteroventral (AVCN), posteroventral (PVCN) and dorsal cochlear nucleus (DCN) at 1, 20 and 90 days after deafferentation. An increase in IGF-1 immunostaining within AVCN, PVCN and DCN was observed ipsilaterally at all survival times after cochlear ablation when compared with the contralateral side and unoperated animals. This increase was accompanied by a significant ipsilateral increase in the mean gray level of synaptophysin immunostaining as well as a decrease in the area of synaptophysin immunostaining at 1 and 20 days after the ablation in AVCN, PVCN and DCN compared with the contralateral side and control animals. These changes in synaptophysin immunostaining were no longer present 90 days after cochlear ablation. The present results provide evidence of a persistent upregulation in IGF-1 and a transitory upregulation in synaptophysin levels in the cochlear nucleus that may reflect neuroprotective mechanisms following the loss of trophic support from spiral ganglion neurons.

In the present study, unilateral cochlear ablations were performed in adult ferrets to evaluate possible time-dependent modifications of synaptophysin and insulin-like growth factor-1 (IGF-1) in the central nucleus of the inferior colliculus (CNIC). Using densitometric analysis, synaptophysin and IGF-1 immunostaining were assessed at 1 (PA1) and 90 (PA90) days after cochlear ablation. The results demonstrated that 1 day after the lesion there was an increase in the levels of synaptophysin immunostaining bilaterally in the CNIC compared to control animals. That increase was no longer present at 90 days after the ablation. Overall levels of IGF-1 immunostaining at PA1 were increased significantly within neurons and neuropil. However, at PA90, only IGF-1 immunostaining contralateral to the lesion was elevated compared to control animals, although elevation was less than that observed at PA1. These results suggest that cochlear ablation appears to affect synaptophysin and IGF-1 protein levels bilaterally in the CNIC.

In the present study, unilateral cochlear ablations were performed in adult ferrets in order to determine whether an upregulation of the calretinin immunostained plexus in the central nucleus of the inferior colliculus occurs and if so, what the time course of this upregulation is. Accordingly, the mean gray level and the calretinin-immunostained area of the axonal plexus in the central nucleus of the inferior colliculus were evaluated at 1, 20 and 90 days after cochlear ablation. In unoperated animals, the calretinin-immunostained plexus was bilaterally symmetric. In ablated animals, both the mean gray level and the immunostained area of the plexus increased in the central nucleus of the inferior colliculus contralateral to the lesion compared with both the ipsilateral side and unoperated animals. This upregulation was present 24 h after the ablation and did not change at the two subsequent time points. In a previous study in young ferrets, the immunostained area of the plexus in the central nucleus of the inferior colliculus contralateral to the lesion increased 200% compared with control ferrets [J Comp Neurol 460 (2003) 585], whereas it increased only 33% in adult ferrets. These findings suggest that 1) calretinin upregulation in the contralateral central nucleus of the inferior colliculus following cochlear ablation occurs by 24 h after cochlear ablation and 2) there is an age-related decline in the magnitude of this upregulation after cochlear ablation.

It is known that the dorsal cochlear nucleus and medial geniculate body in the auditory system receive significant inputs from somatosensory and visual-motor sources, but the purpose of such inputs is not totally understood. Moreover, a direct connection of these structures has not been demonstrated, because it is generally accepted that the inferior colliculus is an obligatory relay for all ascending input. In the present study, we have used auditory neurophysiology, double labeling with anterograde tracers, and retrograde tracers to investigate the ascending projections of the cochlear nuclear complex. We demonstrate that the dorsal cochlear nucleus and the small cell cap of the ventral cochlear nucleus have a direct projection to the medial division of the medial geniculate body. These direct projections from the cochlear nucleus complex bypass the inferior colliculus and are widely distributed within the medial division of the medial geniculate, suggesting that the projection is not topographic. As a nonlemniscal auditory pathway that parallels the conventional auditory lemniscal pathway, its functions may be distinct from the perception of sound. Because this pathway links the parts of the auditory system with prominent nonauditory, multimodal inputs, it may form a neural network through which nonauditory sensory and visual-motor systems may modulate auditory information processing.

The medial nucleus of the trapezoid body (MNTB) is one of three major nuclei of the superior olivary complex and provides an important inhibitory input from the contralateral ear to the lateral superior olivary nucleus (LSO) in the initial binaural pathway for coding interaural intensity differences. The major input to the MNTB from the contralateral anteroventral cochlear nucleus (AVCN) involves giant, calyx-like endings that have a one-to-one relationship with cells in the MNTB as confirmed in the ferret in this study. The main objective of the present study was to define the subsequent organization of projections from cells receiving these calyx-like endings. Several anatomical tracers (Phaseolus vulgaris leucoagglutinin, dextran-biotin, and biocytin) were used that are transported both anterogradely and retrogradely within neuronal projections in order to define the organization of MNTB connections with the LSO in the adult ferret. Analysis focused on determining the topography in both the transverse and longitudinal planes of the projections. Focal tracer injections in the LSO resulted in retrograde labeling of a long, narrow column of cells in the MNTB. The orientation and location of labeled cells was dependent on the medial-lateral position of the injection site. In the rostral-caudal dimension of MNTB, there was no such topographic relation between the injection site and the position of labeled cells. Labeled cells in the MNTB were distributed more or less evenly in a longitudinal column regardless of whether the injection site was restricted to the rostral, middle or caudal part of the LSO. In keeping with this pattern, tracer injections in the MNTB resulted in bands of labeled axons that distributed endings throughout the rostral-caudal axis of the LSO. These bands or sheets varied in medial-lateral position relative to the location of the injection site, but lacked any such rostral-caudal gradient. Thus, overall the MNTB-LSO projections have a convergent-divergent pattern of organization. While MNTB cells receive singular calyx-like endings from the AVCN, LSO cells receive projections from a long column of cells in the MNTB. Implications for processing interaural intensity differences are discussed.

Publisher Summary The chapter discusses the Axon decussation and midline glia in the developing ferret auditory hindbrain. The timing of trapezoid body fiber out growth in relation to the timing of medial superior olive (MSO) and lateral superior olive (LSO) cell migration to the ventral hindbrain, the apparently changing distribution of glycoconjugates in relation to trapezoid body formation, and the distribution of midline glia in the hindbrain. Pioneer fibers from the cochlear nucleus reach the midline before the cells of the superior olivary complex complete migration to the ventral hindbrain. These fibers may be guided to their site of decussation, as are spinal cord commissural neurons, by netrin diffusing from the floor plate. Subsequently, the superior olivary complex begins to release a local chemotropic factor, resulting in the collateralization of cochlear nucleus fibers and projection of those fibers into their target nuclei. As the cochlear nucleus projection in the trapezoid body continues to increase, contralaterally projecting fibers are guided through the midline by a combination of extracellular matrix and glial guidance cues. Inappropriate fibers are prevented from crossing the midline by both the molecular and structural characteristics of the midline glia.

The distribution of glycine-immunopositive cells and axonal endings was studied in the adult and early postnatal ferret superior olive. As in other species, the most prominent glycine-immunopositive cell group in the adult ferret superior olive was the medial nucleus of the trapezoid body. Other darkly immunostained cells were present, although more scattered, in most periolivary regions, including the lateral and ventral trapezoid body nuclei. In the lateral superior olivary nuclei, glycine-immunopositive cells were intermingled with immunonegative cells. A comparable population of cells in the ipsilateral lateral superior olivary nucleus was retrogradely labeled in cases with unilateral injections of tritiated glycine in the inferior colliculus. Glycine-immunopositive puncta were widely distributed in the neuropil in most periolivary regions, including dense accumulations in the dorsomedial periolivary region and ventral and lateral nuclei of the trapezoid body. In the lateral and medial superior olivary nuclei, immunopositive puncta were distributed around the principal cells in characteristic perisomatic halos. In postnatal ferrets, immunopositive cell bodies were first observed by postnatal day 7 and were distributed in regions comparable to regions in the adult, with the exception that immunopositive cells in the lateral superior olivary nucleus did not appear until about postnatal day 28. There was diffuse staining in the neuropil in principal and periolivary nuclei by postnatal day 7. During the third postnatal week, the immunostaining in the neuropil began to take on a more granular appearance and immunopositive puncta could be seen by postnatal day 35. In the lateral and medial superior olivary nuclei, the earliest distribution of immunostaining in the neuropil was nonuniform, being greater in the high-frequency, medial, and ventral regions, respectively. The density gradient in these areas was gradually eliminated over the next 2 postnatal weeks as immunostained processes and endings appeared over greater portions of the nuclei. © 1995 Wiley-Liss, Inc.

The laterality of projections from the lateral superior olivary nucleus (LSO) to the inferior colliculus was studied in adult and immature postnatal ferrets. In the adult ferret, large unilateral injections of horseradish peroxidase (HRP) in the inferior colliculus labeled about equal proportions of cells in the ipsilateral and contralateral lateral superior olivary nuclei. The contralateral labeled cells consistently were more densely labeled than those on the ipsilateral side. Double labeling experiments using fluorescent dyes indicated that only about 3% of LSO cells in the adult give rise to collaterals ending in the inferior colliculus on both sides. As expected, the distribution of labeled cells varied topographically in the LSO as a function of the injection site in the inferior colliculus. Dorsolateral inferior collicular injections labeled cells in the lateral limb of the LSO, whereas ventromedial injections labeled cells in the medial limb of the LSO. The proportion of ipsilateral and contralateral labeled cells also varied across the lateral-medial axis of the LSO in some cases. A gradient in laterality was observed in these cases with the lateral limb of the LSO containing the highest proportion of contralateral labeled cells, and the medial limb, the highest proportion of ipsilateral labeled cells. Larger inferior collicular injections resulted in greater proportions of ipsilateral labeling in LSO than smaller injections. Finally, ipsilateral labeled cells tended to be in the marginal region of the LSO, whereas contralateral labeled cells were more common within the core region of the LSO, irrespective of the location along the lateral-medial axis of LSO. The contralateral predominance of labeled cells, greater density of labeling in contralateral cells, different topographic distribution, and regional segregation of ipsilateral and contralateral labeled cells were typical of the LSO in ferret kits by birth, one month before the onset of hearing. Nevertheless, the relative proportion of ipsilateral and contralateral projection cells appears to change during postnatal development. © 1993 Wiley-Liss, Inc.

The dendritic morphology of cells in the lateral superior olivary nucleus was studied with the Golgi method in adult and postnatal ferrets. The lateral superior olivary nucleus in the adult ferret is a convoluted structure with an M-shape in frontal sections. The major cell type appears to have disk-shaped dendritic trees. Most dendritic trees appear to be approximately orthogonal to the curved medial-lateral axis of the nucleus. Depending on their position in the limb and on the plane of section with respect to the dendritic tree, the disk-shaped cells are either bipolar or radiate in orientation. One subclass of disk-shaped cells has secondary dendritic branches that end as tufts of tendril-like processes. In a second subclass of cells, the dendrites exhibit several orders of dichotomous branching and lack obvious tufts of terminal processes. Marginal cells are observed at the border of the nucleus and have dendrites restricted to the margins of the cell plate. The bipolar orientation of disk-shaped cells orthogonal to the axis of the limbs is already apparent by the time of birth. Transient spines and other appendages are abundant on somata and dendrites during the first postnatal week. By the end of the first postnatal month only distal appendages are found. Tufts of fine tendril-like processes appear at the ends of dendrites between postnatal days 28 and 56.

Dendritic morphology and development in the medial superior olivary nucleus of the ferret were studied using the Golgi method. In the adult ferret most medial superior olivary neurons had disk-shaped dendritic fields. These dendritic fields were oriented such that cells in a coronal plane of section appeared bipolar with major lateral and medial dendritic axes. In the horizontal plane the dendrites radiated about the soma. Dendrites of principal cells branched distally into tufts of numerous, tertiary processes that were beaded and thin. Peripheral cells in the fiber mantle encircling the nucleus were generally spindle-shaped or tripolar and lacked the tufted dendrites of principal cells. The dendrites of these peripheral cells coursed parallel to the nucleus both dorsoventrally and rostrocaudally. Horizontally oriented dendrites were observed even at birth for some cells in the medial superior olivary nucleus and bipolar dendritic fields were typical of most cells by the end of the second postnatal week. Dendrites of immature cells varied in caliber and radiated in all directions from the soma. Around postnatal days 8-10 transient appendages appeared on the soma and dendrites, first proximally and then more distally. These appendages persisted until the first postnatal month. Cell size and dendritic radius increased markedly during this same period. Postnatal days 28-30 were marked by the first appearance of tufts of tertiary dendritic branches. The tendril-like processes continued to increase in length until about the end of the second postnatal month.

Recent neurophysiological experiments have implicated the vestibular complex of mammals as a relay site for non-vestibular sensory information. In particular, it has been demonstrated that both neck proprioceptive and visual stimulation can modify the activity of secondary vestibular neuronslS, 20. The vestibulocerebellum might be an indirect source of neck proprioceptive and visual information to the vestibular nuclei since the activity of floccular and nodular Purkinje cells is modified by these sensory modalities and since the vestibulocerebellum projects onto the vestibular nucleiZ,4,2L However, neck proprioceptive information also ascends to the vestibular nuclei via a more direct spinal pathway 11. We have investigated the contribution of the vestibulocerebellum to the visual modulation of the activity of neurons in the vestibular complex. Neither dorsal cap lesions nor total cerebellectomies abolish the visual modulation of secondary vestibular neuronal activity 12 (Barmack and Pettorossi, unpublished observations). These findings are in agreement with behavioral observations which stress that damage to the vestibular nuclei causes a greater impairment of optokinetic reflexes than does cerebellectomy3, s. Therefore, using the HRP tracer technique, we have undertaken an investigation of alternative pathways by which visual information could reach the vestibular nuclei. Eight albino and Dutch-belted rabbits were used for unilateral, and in two cases, bilateral injections of HRP into the vestibular complex. The results from the two species were equivalent. The injection sites were localized by physiological techniques. Extracellular microelectrode recordings from the vestibular nuclei were obtained with a tungsten microelectrode while the rabbit was rotated in the horizontal or vertical planes in a bi-axial rate table. Single medial vestibular neurons were identified by their increased rate of discharge in phase with ipsilateral velocity of the rate table, and by their increased rate of discharge evoked by posterior-anterior optokinetic stimulation of the ipsilateral eye. Following physiological identification of the medial vestibular nucleus, the tungsten microelectrode was withdrawn and replaced with a glass micropipette containing 25~ HRP (Sigma IX). Its position was confirmed by observing multiple-unit activity during horizontal oscillation of the rate table. Pressure

Previous studies suggest that the principal cells of the medial nucleus of the trapezoid body (MNTB) give rise to the projection from MNTB to the lateral superior olivary nucleus (LSO) of the same side, where they mediate rapid inhibitory effects of contralateral sound stimulation. In the present study, we explored certain morphological features of this connection as well as several other projections of the MNTB by using anterograde and retrograde axonal tracing methods. Following injections of tritiated leucine into MNTB, labeled axons reached LSO by passing ventral to, dorsal to, and through the medial superior olivary nucleus, and gave rise to labeling around the somata and proximal dendrites of LSO fusiform cells. As measured in autoradiograms of 2 micron plastic sections, these axons had a modal diameter of 5-6 micron. Terminal labeling, tentatively attributed to principal cell axons, was also seen in the ventral nucleus of the lateral lemniscus (VNLL) and the dorsomedial and ventromedial periolivary nuclei. HRP injections into the LSO and the VNLL showed that the principal cell projected to both of these nuclei and revealed a topographic arrangement of the projection to the LSO which is consistent with tonotopic maps determined electrophysiologically. Control HRP injections demonstrated that other minor projections of the MNTB arose from minor cell populations in this nucleus. The findings provide a morphological correlate of certain physiological findings and suggest a wider role for the MNTB in the ascending auditory system than previously has been supposed.

A comprehensive search for subcortical projections to the cat superior colliculus was conducted using the retrograde horseradish peroxidase (HRP) method. Over 40 different subcortical structures project to the superior colliculus. The more notable among these are grouped under the following categories. Visual structures: ventral lateral geniculate nucleus, parabigeminal nucleus, pretectal area (nucleus of the optic tract, posterior pretectal nucleus, nuclei of the posterior commissure). Auditory structures: inferior colliculus (external and pericentral nuclei), dorsomedial periolivary nucleus, nuclei of the trapezoid body, ventral nucleus of the lateral lemniscus. Somatosensory structures: sensory trigeminal complex (all divisions, but mainly the γ division of nucleus oralis), dorsal column nuclei (mostly cuneate nucleus), and the lateral cervical nucleus. Catecholamine nuclei: locus coeruleus, raphe dorsalis, and the parabrachial nuclei. Cerebellum: medial, interposed, and lateral nuclei, and the perihypoglossal nuclei. Reticular areas: zona incerta, substantia nigra, midbrain tegmentum, nucleus paragigantocellularis lateralis, and the hypothalamus. Evidence is presented that only the parabigeminal nucleus, the nucleus of the optic tract, and the posterior pretectal nucleus project to the superficial collicular layers (striatum griseum superficiale and stratum opticum), while all other afferents terminate in the deeper layers of the colliculus. Also presented is information concerning the rostrocaudal distribution of some of these afferent connections. These findings stress the multiplicity and diversity of inputs to the deeper collicular layers, and more specifically, identify multiple sources of the physiologically well-known representations of the somatic and auditory modalities in the colliculus.

Subdivisions of the cochlear nuclear complex give rise to a number of discrete projections to certain cell groups of the superior olivary complex and also received substantial descending projections from the periolivary nuclei. In the present study, we sought to determine by means of retrograde transport of horseradish peroxidase (HRP), and anterograde transport of radiolabeled protein, if the periolivary nuclei give rise to discrete projections to the various subdivisions of the cochlear nuclear complex. Following medium to large injections of HRP into the cochlear nucleus, irrespective of location, labeled cells were found in all periolivary nuclei bilaterally. In every case more than 40% of the labeled cells were found in the lateral nucleus of the trapezoid body on the same side and the ventral nucleus of the trapezoid body of both sides. Other periolivary nuclei contributing more than 5% of the total number of cells in individual cases were the contralateral lateral nucleus of the trapezoid body and the ipsilateral anterolateral and dorsal periolivary nuclei. Injections of tritiated leucine into periolivary nuclei gave rise to axonal labeling to the trapezoid body and the dorsal acoustic stria, usually bilaterally, and to terminal labeling that was widely distributed within the cochlear nuclear complex. In several cases with small injections, particularly in the lateral nucleus of the trapezoid body, the projections from the periolivary nuclei to the anteroventral and dorsal cochlear nuclei connected areas described as having similar best-frequency representation. The autoradiographic data corroborated the main results from the HRP experiments and provided additional information permitting these conclusions: (1) the projections from the periolivary nuclei to the cochlear nuclear complex are organized tonotopically, at least in part; (2) each periolivary nucleus (and perhaps individual cells), projects widely throughout the cochlear nuclear complex; (3) the pattern of termination of projections from different periolivary nuclei to a given region of the cochlear nuclear complex are similar, as seen in autoradiograms, and (4) the lateral and dorsal periolivary nuclei project mainly ipsilaterally, while the medial periolivary nuclei project bilaterally with a contralateral bias. The magnitude of these projections and their widespread distribution within the cochlear nuclear complex would suggest an important role for the descending projections in the normal functioning of the cochlear nucleus.

This study examines the dorsal nucleus of the lateral lemniscus (DNLL) and its afferent and efferent connections. In Nissl-stained material, DNLL has three parts: dorsal, ventral, and lateral. Although each part contains neurons with similar Nissl patterns, the subdivisions may be distinguished by the size, shape, and orientation of the cells. The lateral DNLL contains a mixture of DNLL neurons and cells from the sagulum. Afferent connections to DNLL were investigated with anterograde axonal transport techniques. Bilateral inputs to DNLL arise from the anteroventral cochlear nucleus and lateral superior olive, while unilateral inputs are provided by the ipsilateral medial superior olive and the contralateral DNLL. The inputs appear to have a tonotopic organization. Afferent fibers to DNLL form horizontal bands that are continuous both mediolaterally and rostrocaudally. All parts of DNLL do not share the same inputs, and a medial-to-lateral gradient in the labeling of some pathways is evident. To study the efferent connections of DNLL, both retrograde and anterograde axonal transport techniques were used. The DNLL projects to the inferior colliculus and the contralateral DNLL. The topography of these projections suggests that areas of similar tonotopic organization are connected. In the inferior colliculus, the projection is heaviest to the central nucleus and extends to the adjacent dorsal and caudal cortex, the rostral pole nucleus, and the ventrolateral nucleus. Axons from DNLL terminate along the fibrodendritic laminae of the central nucleus as bands that are prominent on the contralateral side, whereas those on the ipsilateral colliculus are more diffuse. The afferent and efferent connections of DNLL constitute a multisynaptic pathway, parallel to the other ascending pathways to the inferior colliculus. The other ascending pathways include the direct pathways from the cochlear nucleus to the inferior colliculus and the indirect pathways via the superior olivary complex. Ascending pathways are discussed as to their relationship to the subdivisions of the inferior colliculus, the laterality of their projections, and their banding patterns in the central nucleus. In contrast to the excitatory pathways to the inferior colliculus, the neurons in DNLL may use GABA as a neurotransmitter. Axons from the DNLL terminate in the inferior colliculus as bands that could have a unique inhibitory function. Thus, the multisynaptic, DNLL pathway may provide feed-forward inhibitory inputs to the inferior colliculus, bilaterally, and to the contralateral DNLL.

In this study we investigated the collateral axonal projections to the superior olivary complex using the combined anterograde and retrograde transport of wheat-germ agglutinin conjugated with horseradish peroxidase. Small injections of this tracer were placed in the lateral or medial superior olivary nuclei in cats, and the location of anterograde label in the alternate nuclei of the superior olivary complex was determined. Injections of [3H]leucine were also placed in these nuclei for control purposes. After wheat-germ agglutinin-horseradish peroxidase injections in the lateral superior olivary nucleus anterograde label was observed bilaterally in the medial superior olivary nuclei. Likewise, after injections in the medial superior olivary nucleus anterograde label was observed in the contralateral medial and lateral superior olivary nuclei. The topography of the anterograde label was always precise and varied predictably as a function of the injection site. Most retrogradely labeled cells were located in the ipsilateral anteroventral cochlear nucleus and the medial nucleus of the trapezoid body. Various interpretations of the data are considered. Our primary conclusion is that cells in the anteroventral cochlear nucleus are a major source of collaterals to both the ipsilateral and contralateral nuclei of the superior olivary complex.

In this study the organization of the projection from the lateral superior olivary nucleus (LSO) to the inferior colliculus was investigated in the cat by using anterograde tract-tracing techniques. The findings indicated that LSO projected bilaterally to the central nucleus of the inferior colliculus as well as to the ventrolateral and rostral pole nuclei. In the central nucleus a larger medial component of the projection ended in pars medialis and centralis. A smaller lateral component ended in the region of the pars lateralis. Both components of the projection appeared to be topographically organized, but in the lateral component the low-frequency part of LSO appeared to have greater representation. The uncrossed and crossed LSO projections to the inferior colliculus exhibited several important differences in their distribution. First, periodic bands of dense labeling were more prominent in the distribution of the uncrossed projection. The bands measured 150–200 μ in thickness and in some cases interruptions or gaps were present along the length of the bands. The distribution of the crossed projection was more diffuse, but some banding was also apparent. Second, the positions of the bands of dense labeling on the two sides were not homotopic as determined by labeling projections from the ipsilateral and contralateral LSO in the same tissue. The dense bands labeled with WGA-HRP from an injection in LSO on one side and bands labeled with 3H-leucine from an injection in LSO on the other side either were interdigitating or were only partially overlapping. Finally, the area over which the uncrossed projection distributed endings varied in size with respect to that of the crossed projection. The variation in size of the area of the projections was a function of the frequency representation. A model based on the three-dimensional reconstruction of bands as projection sheets is proposed as a substrate for selective integration of afferents in the inferior colliculus.

The localization of the motor neurons to the tensor tympani (TT) muscle was studied using the method of retrograde transport of horseradish peroxidase (HRP). After intramuscular injections of HRP, specifically labeled neurons were found in the ventral, parvocellular portion of the trigeminal motor nucleus. These cells had a medial relation to the rootlets of the trigeminal nerve and, rostrally, to the lateral lemniscus. The results are compared to those of other investigators and a generalization is suggested for the localization of these neurons which allows for species variation. Other incidental findings are also discussed.

Connections of a posteromedial region of the ventral nucleus of the lateral lemniscus were examined in the cat using the autoradiographic tracing method. This sub-collicular region previously had been shown, using retrograde transport of horseradish peroxidase, to send axons to the superior colliculus 10 . The autoradiographic findings revealed that many axons from the posteromedial region of the ventral nucleus of the lateral lemniscus that entered the superior colliculus continued into the midbrain reticular formation. Moreover, other axons traced rostral to the inferior colliculus into the thalamus ended in the medial geniculate nucleus, bilaterally. Experiments in which horseradish peroxidase was placed in the medial geniculate nucleus retrogradely labeled the large neurons in the posteromedial region supporting the autoradiographic observations. Other sub-collicular regions also contained labeled cells in these cases, including the main body of the ventral nucleus of the lateral lemnicus and scattered cell groups around the superior olivary complex.

The topographic distribution of projections from the ventral nucleus of the lateral lemniscus (VNLL) in the cat was investigated with the autoradiographic tracing method. The origin of minor projections was verified by retrograde tracing methods. Small injections of tritiated leucine were placed in restricted zones of VNLL. A major afferent fiber system to the inferior colliculus was labeled in all cases. From the injection site labeled fibers coursed through and around the nuclei of the lateral lemniscus to enter the ipsilateral inferior colliculus. Regardless of the position or small size of the injection, labeled fibers distributed widely in the inferior colliculus. Fibers ended in the central nucleus and deeper layers of the dorsal cortex in most cases. There was also labeling in the ventrolateral nucleus, but very few fibers ended as lateral as the lateral nucleus. A small number of labeled fibers passed from the inferior colliculus into the nucleus of the brachium of the inferior colliculus and adjacent tegmental areas. Some labeled fibers entered the commissure of the inferior colliculus where they were traced into the dorsal cortex and rostral pole of the inferior colliculus on the side contralateral to the injection site. Though the projections labeled in individual cases were similar in their divergent pattern within the central nucleus of the inferior colliculus, specific variations in the pattern were found. The dorsal zone of VNLL projected more heavily to the deeper layers of the dorsal cortex and an adjacent field in the central nucleus than the other zones. Dorsal injections in the middle zone of VNLL, on the other hand, labeled the medial part of the central nucleus more heavily, whereas ventral injections in the middle zone resulted in heavier lateral labeling. The ventral zone of VNLL projected heavily to a central field in the central nucleus. In addition to this major afferent system of VNLL to the inferior colliculus, a smaller descending projection was found. The descending projection ended mainly in the dorsomedial periolivary region and ventral nucleus of the trapezoid body. However, in some cases a few fibers were traced to the cochlear nuclei. Finally, we observed projections to the medial geniculate body from the dorsal and ventral zones of VNLL that ended diffusely in the medial division of the medial geniculate body. Possibly some fibers from the dorsal zone contribute to a broader projection of the lateral tegmentum to the dorsal division of the medial geniculate body.

Possible anatomical pathways mediating superior colliculus control of pinna movements were determined in the cat using the orthograde autoradiographic tracing method and the retrograde horseradish peroxidase technique. This was done in the following manner. First, the division of the facial nucleus that innervates the pinna muscles was determined by injecting the pinna muscles with HRP and surveying the facial nucleus for retrogradely filled cells. Second, the brainstem regions that project the facial nucleus were identified using the horseradish peroxidase method. Third, the superior colliculus projections to these areas were studied using the autoradiographic tracing method. The results suggest that superior colliculus control of pinna movements is mediated entirely by indirect connections with the facial nucleus and that these connections occur mainly in a paralemniscal zone in the lateral midbrain. Of all the brainstem regions shown by the horseradish peroxidase experiments to project to the facial nucleus only this midbrain paralemniscal zone received a projection from the superior colliculus that was dense and overlapped precisely the region containing facial projecting neurons. Further autoradiographic tracing revealed that the facial nucleus was the primary brainstem target of this paralemniscal zone and that all paralemniscal fibers projecting to the facial nucleus ended in the subdivision that innervates the pinna muscles. Other paralemniscal efferents terminate in the opposite paralemniscal zone. The data suggest that other connections between the superior colliculus and the facial nucleus may occur in the cuneiform nucleus of the midbrain, the region around the oculomotor complex, and the reticular formation dorsal to the superior olive.