Effects of acoustic trauma on the auditory system of the rat: The role of microglia.

Exposure to loud, prolonged sounds (acoustic trauma, AT) leads to the death of both inner and outer hair cells (IHCs and OHCs), death of neurons of the spiral ganglion and degeneration of the auditory nerve. The auditory nerve (8cn) projects to the three subdivisions of the cochlear nuclei (CN), the dorsal cochlear nucleus (DC) and the anterior (VCA) and posterior (VCP) subdivisions of the ventral cochlear nucleus (VCN). There is both anatomical and physiological evidence for plastic reorganization in the denervated CN after AT. Anatomical findings show axonal sprouting and synaptogenesis; physiologically there is an increase in spontaneous activity suggesting reorganization of circuitry. The mechanisms underlying this plasticity are not understood. Recent data suggest that activated microglia may have a role in facilitating plastic reorganization in addition to removing trauma-induced debris. In order to investigate the roles of activated microglia in the CN subsequent to AT we exposed animals to bilateral noise sufficient to cause massive hair cell death. We studied four groups of animals at different survival times: 30 days, 60 days, 6 months and 9 months. We used silver staining to examine the time course and pattern of auditory nerve degeneration, and immunohistochemistry to label activated microglia in the denervated CN. We found both degenerating auditory nerve fibers and activated microglia in the CN at 30 and 60 days and 6 months after AT. There was close geographic overlap between the degenerating fibers and activated microglia, consistent with a scavenger role for activated microglia. At the longest survival time, there were still silver-stained fibers but very little staining of activated microglia in overlapping regions. There were, however, activated microglia in the surrounding brainstem and cerebellar white matter.

Dissociation of doublecortin expression and neurogenesis in unipolar brush cells in the vestibulocerebellum and dorsal cochlear nucleus of the adult rat.

We have previously shown expression of the protein doublecortin (DCX) in unipolar brush cells (UBCs) in the dorsal cochlear nucleus and vestibulocerebellum of the adult rat. We also saw DCX-immunoreactive elements with the appearance of neuroblasts around the fourth ventricle. Expression of DCX is seen in newborn and migrating neurons and hence considered a correlate of neurogenesis. There were two interpretations of the expression of DCX in UBCs. One possibility is that there might be adult neurogenesis of this cell population. Adult neurogenesis is now well-established, but only for the dentate gyrus of the hippocampus and the subventricular zone. The other possibility is that there is prolonged expression of DCX in adult UBCs that may signal a unique role in plasticity of these neurons. We tested the neurogenesis hypothesis by systemic injections of bromodeoxyuridine (BrdU), a thymidine analog, followed by immunohistochemistry to examine the numbers and locations of dividing cells. We used several different injection paradigms, varying the dose of BrdU, the number of injections and the survival time to assess the possibility of neuronal birth and migration. We saw BrdU-labeled cells in the cerebellum and brainstem; cell division in these regions was confirmed by immunohistochemistry for the protein Ki67. However, neither the numbers nor the distribution of labeled nuclei support the idea of adult neurogenesis and migration of UBCs. The function of DCX expression in UBC's in the adult remains to be understood.

Expression of doublecortin, a neuronal migration protein, in unipolar brush cells of the vestibulocerebellum and dorsal cochlear nucleus of the adult rat.

Doublecortin (DCX) is a microtubule-associated protein that is critical for neuronal migration and the development of the cerebral cortex. In the adult, it is expressed in newborn neurons in the subventricular and subgranular zones, but not in the mature neurons of the cerebral cortex. By contrast, neurogenesis and neuronal migration of cells in the cerebellum continue into early postnatal life; migration of one class of cerebellar interneuron, unipolar brush cells (UBCs), may continue into adulthood. To explore the possibility of continued neuronal migration in the adult cerebellum, closely spaced sections through the brainstem and cerebellum of adult (3-16 months old) Sprague-Dawley rats were immunolabeled for DCX. Neurons immunoreactive (ir) to DCX were present in the granular cell layer of the vestibulocerebellum, most densely in the transition zone (tz), the region between the flocculus (FL) and ventral paraflocculus (PFL), as well as in the dorsal cochlear nucleus (DCN). These DCX-ir cells had the morphological appearance of UBCs with oval somata and a single dendrite ending in a brush. There were many examples of colocalization of DCX with Eps8 or calretinin, UBC markers. We also identified DCX-ir elements along the fourth ventricle and its lateral recess that had labeled somata but lacked the dendritic structure characteristic of UBCs. Labeled UBCs were seen in nearby white matter. These results suggest that there may be continued neurogenesis and/or migration of UBCs in the adult. Another possibility is that UBCs maintain DCX expression even after migration and maturation, reflecting a role of DCX in adult neuronal plasticity in addition to a developmental role in migration.

Type B GABA receptors contribute to the restoration of balance during vestibular compensation in mice.

Following unilateral vestibular damage (UVD), vestibular compensation restores both static and dynamic vestibular reflexes. The cerebellar cortex provides powerful GABAergic inhibitory input to the vestibular nuclei which is necessary for compensation. Metabotropic GABA type B (GABA(B)) receptors in the vestibular nuclei are thought to be involved. However, the contribution of GABA(B) receptors may differ between static and dynamic compensation. We tested static and dynamic postural reflexes and gait in young mice, while they compensated for UVD caused by injection of air into the vestibular labyrinth. The effects of an agonist (baclofen), an antagonist (CGP56433A) and a positive allosteric modulator (CGP7930) of the GABA(B) receptor were evaluated during compensation. Static postural reflexes recovered very rapidly in our model, and baclofen slightly accelerated recovery. However, CGP56433A significantly impaired static compensation. Dynamic reflexes were evaluated by balance-beam performance and by gait; both showed significant decrements following UVD and performance improved over the next 2 days. Both CGP56433A and baclofen temporarily impaired the ability to walk on a balance beam after UVD. Two days later, there were no longer any significant effects of drug treatments on balance-beam performance. Baclofen slightly accelerated the recovery of stride length on a flat surface, but CGP7930 worsened the gait impairment following UVD. Using immunohistochemistry, we confirmed that GABA(B) receptors are abundantly expressed on the vestibulospinal neurons of Deiters in mice. Our results suggest that GABA(B) receptors contribute to the compensation of static vestibular reflexes following unilateral peripheral damage. We also conclude that impairment of the first stage of compensation, static recovery, does not necessarily result in an impairment of dynamic recovery in the long term.

The human dentate nucleus: a complex shape untangled.

The dentate nucleus is the largest single structure linking the cerebellum to the rest of the brain. The peculiar shape and large size of the human dentate nucleus have sparked a number of theories about the role of the cerebellum in human evolution. Some of the proposed ideas could be explored by comparative studies of humans and apes, but comparative studies are hindered because of the complex three dimensional shape of the human dentate. Here we present a 3D model based on a quantitative reconstruction of the human dentate; this model can facilitate comparative studies. The dentate nucleus has been partitioned into dorsal and ventral lamellae based on sheet thickness. Our data show that the thicker ventral lamella occupies a distinctly smaller portion of the human dentate than previously hypothesized. Within the dorsal lamella there is a medial to lateral increase in depth of dentate folds. However, the dorsal lamella retains a thin sheet thickness unlike the macrogyric ventral lamella, in which sheet thickness is increased. The appearance of larger folds laterally reflects the emergence of secondary folds that could encompass the projection of the cerebellar hemispheres, minimizing convergence of different corticonuclear microzones. Thus, the unique feature of the hominoid dentate is the development of a large surface area and an expansion of its mediolateral width. We propose that this is to allow for a large number of independent corticonuclear modules that can modulate an equal large number of sequential motor acts.

Distribution of corticotectal cells in macaque.

We compared the cortical inputs to the superficial and deep compartments of the superior colliculus, asking if the corticotectal system, like the colliculus itself, consists of two functional divisions: visual and visuomotor. We made injections of retrograde tracer extending into both superficial and deep layers in three colliculi: the injection site involved mainly the upper quadrant representation in one case, the lower quadrant representation in a second case, and both quadrants in a third. In a fourth colliculus, the tracer injection was restricted to the lower quadrant representation of the superficial layers. After injections involving both superficial and deep layers, labeled cells were seen over V1, many prestriate visual areas, and in prefrontal and posterior parietal cortex. Both the density of labeled cells and the degree of visuotopic order as inferred from the distribution of labeled cells in cortex varied among areas. In visual areas comprising the lower levels of the cortical hierarchy, visuotopy was preserved, whereas in "higher" areas the distribution of labeled cells did not strongly reflect the visuotopic location of the injection. Despite the widespread distribution of labeled cells, there were several areas with few or no labeled cells: MSTd, 7a, VIP, MIP, and TE. In the case with an injection restricted to superficial layers, labeled cells were seen only in V1 and in striate-recipient areas V2, V3, and MT. The results are consistent with the idea that the corticotectal system consists of two largely nonoverlapping components: a visual component consisting of striate cortex and striate-recipient areas, which projects only to the superficial layers, and a visuomotor component consisting of many other prestriate visual areas as well as frontal and parietal visuomotor areas, which projects to the deep compartment of the colliculus.

Vertical meridian representation on the prelunate gyrus in area V4 of macaque.

The representation of the lower quadrant in area V4 is presently thought to extend along the prelunate gyrus from a foveal representation laterally all the way to the dorsal end of the superior temporal sulcus. However, several studies suggest the possibility of a more complex organization. To see if the visuotopic organization on the crown of the gyrus was relatively homogeneous or instead contained inhomogeneities indicative of more complex organization, we recorded from a grid of points over the prelunate gyrus. Receptive-field size and scatter in the region are large, making it difficult to infer topography from simple inspection of receptive-field sequences. We developed an averaging procedure using data from all recording sites to detect an inhomogeneity in topography with respect to the vertical meridian. With this procedure, we found a vertical meridian representation just medial to the medial end of the lateral sulcus. We also found a significant difference in the incidence of orientation sensitivity on either side of the meridian representation. The results show that the crown of the prelunate gyrus cannot be described as a single homogeneous region, but instead contains at least two different sub-regions adjoining along a shared representation of the vertical meridian.

Serotonergic innervation of the primate claustrum.

The claustrum is reciprocally and topographically connected with all functional areas of the cerebral cortex. Different cortical areas differ in the source, density, and laminar distribution of serotonergic innervation, with visual cortex receiving an especially rich serotonergic innervation. We asked if there were likewise differences in serotonergic innervation in different regions of the claustrum. We analyzed 50-microm coronal sections through the claustrum of the macaque monkey processed using standard immunohistochemical techniques and an antibody to serotonin. We found labeled fibers throughout the dorsal-ventral and anterior-posterior extent of the claustrum. A few fibers were relatively straight and lacked varicosities. Most fibers had varicosities; the size, shape, and spacing of varicosities varied among fibers and even along a single fiber. Some stained fibers partially encircled cells, and varicosities were seen in close apposition to the cell bodies. There was a major difference between dorsal and ventral claustrum in the pattern of stained fibers. In the ventral, visual, claustrum, stained segments of axons were short and randomly arranged relative to each other, and there were many stained puncta. In the more dorsal, nonvisual claustrum, many fibers ran in a dorsal-ventral direction, along the long axis of the claustrum, and could be followed for long distances.

Cerebellar lesions and prism adaptation in macaque monkeys.

If a laterally displacing prism is placed in front of one eye of a person or monkey with the other eye occluded, they initially will point to one side of a target that is located directly in front of them. Normally, people and monkeys adapt easily to the displaced vision and correct their aim after a few trials. If the prism then is removed, there is a postadaptation shift in which the subject misses the target and points in the opposite direction for a few trials. We tested five Macaque monkeys for their ability to adapt to a laterally displacing prism and to show the expected postadaptation shift. When tested as normals, all five animals showed the typical pattern of adaptation and postadaptation shift. Like human subjects, the monkeys also showed complete interocular transfer of the adaptation but no transfer of the adaptation between the two arms. When preoperative training and testing was complete, we made lesions of various target areas on the cerebellar cortex. A cerebellar lesion that included the dorsal paraflocculus and uvula abolished completely the normal prism adaptation for the arm ipsilateral to the lesion in one of the five monkeys. The other four animals retained the ability to prism-adapt normally and showed the expected postadaptation shift. In the one case in which the lesion abolished prism adaptation, the damage included Crus I and II, paramedian lobule and the dorsal paraflocculus of the cerebellar hemispheres as well as lobule IX, of the vermis. Thus in this case, the lesion included virtually all the cerebellar cortex that receives mossy-fiber visual information relayed via the pontine nuclei from the cerebral cortex. The other four animals had damage to lobule V, the classical anterior lobe arm area and/or vermian lobules VI/VII, the oculomotor region. When tested postoperatively, some of these animals showed a degree of ataxia equivalent to that of the case in which prism adaptation was affected, but prism adaptation and the postadaptation shift remained normal. We conclude that in addition to its role in long-term motor learning and reflex adaptation, the region of the cerebellum that was ablated also may be a critical site for a short-term motor memory. Prism adaptation seems to involve a region of the cerebellum that receives a mossy-fiber visual error signal and probably a corollary discharge of the movement.

Immunoreactivity for calcium-binding proteins in the claustrum of the monkey.

The claustrum is topographically and reciprocally connected with many different cortical areas, and anatomical and physiological data suggest it is composed of functionally distinct subdivisions. We asked if the distribution of cells immunoreactive for three calcium-binding proteins, parvalbumin, calbindin D-28k and calretinin would delineate functional subdivisions in the claustrum. We also asked if, as in cortex, different cell types were immunoreactive for the different proteins. We found that cells with parvalbumin-ir were large, multipolar cells. Cells immunoreactive for calretinin were bipolar cells with elongated cell bodies and beaded dendrites. There were three different types of cells immunoreactive for calbindin. The most numerous were small cells with round or oval cell bodies and numerous fine, winding processes. A second type were large multipolar, cells that resembled the parvalbumin-ir cells. The third class were bipolar cells with large, elongated cell bodies. Each type of cell resembles a cell type described in earlier Golgi studies, and each has a morphological cortical counterpart. While the different cell types varied in density, each was seen over the anterior-posterior and dorsal-ventral extent of the claustrum.

Projections from the claustrum to the prelunate gyrus in the monkey.

In order to determine whether the cells in the monkey claustrum which project to visual area V4 are found in the same territory as cells projecting to other visual areas, we made injections of the retrograde fluorescent tracer diamidino yellow into area V4 in two monkeys. Injections of a second tracer, fast blue, were also made in area PM, an area just medial to area V4 on the prelunate gyrus, in one animal. Area V4 injections labeled cells in ventral claustrum over about 5-6 mm of its anterior-posterior extent. The more medial prelunate injection labeled cells in adjacent dorsal and more lateral claustrum. These results, together with data from other studies, suggest that in the monkey, as in the cat, there is a "visual" region of claustrum that is interconnected with multiple visual areas including V1, V2, V4, MT, FST, MST, TEO and TE. The data also suggest that dorsal and lateral to this region is another zone which is connected with different visual areas, including several in posterior parietal cortex.

Comparison of subcortical connections of inferior temporal and posterior parietal cortex in monkeys.

To investigate the subcortical connections of the object vision and spatial vision cortical processing pathways, we injected the inferior temporal and posterior parietal cortex of six Rhesus monkeys with retrograde or anterograde tracers. The temporal injections included area TE on the lateral surface of the hemisphere and adjacent portions of area TEO. The parietal injections covered the posterior bank of the intraparietal sulcus, including areas VIP and LIP. Our results indicate that several structures project to both the temporal and parietal cortex, including the medial and lateral pulvinar, claustrum, and nucleus basalis. However, the cells in both the pulvinar and claustrum that project to the two systems are mainly located in different parts of those structures, as are the terminals which arise from the temporal and parietal cortex. Likewise, the projections from the temporal and parietal cortex to the caudate nucleus and putamen are largely segregated. Finally, we found projections to the pons and superior colliculus from parietal but not temporal cortex, whereas we found the lateral basal and medial basal nuclei of the amygdala to be reciprocally connected with temporal but not parietal cortex. Thus, the results show that, like the cortical connections of the two visual processing systems, the subcortical connections are remarkably segregated.

Bilateral projections from the parabigeminal nucleus to the superior colliculus in monkey.

We examined the distribution of labeled neurons in the parabigeminal nucleus of the monkey following injections of retrograde fluorescent tracers into the superior colliculus. The extent of the visual field representation included in the injection site was assessed from the location of labeled cells in striate cortex. The results suggest a rough topographic organization of the parabigeminal nucleus, with the lower quadrant represented anteriorly and the upper quadrant posteriorly. We also found bilateral projections from the parabigeminal nucleus to both superior colliculi, but the crossed projection appeared to terminate only in that part of the colliculus where the vertical meridian is represented. Parabigeminal cells with a crossed projection were larger than those projecting to the ipsilateral colliculus. The results suggest that the organization of the monkey's parabigemino-tectal system is fundamentally similar to that of many other vertebrates.

Organization of visual inputs to the inferior temporal and posterior parietal cortex in macaques.

It has been proposed that visual information in the extrastriate cortex is conveyed along 2 major processing pathways, a "dorsal" pathway directed to the posterior parietal cortex, underlying spatial vision, and a "ventral" pathway directed to the inferior temporal cortex, underlying object vision. To determine the relative distributions of cells projecting to the 2 pathways, we injected the posterior parietal and inferior temporal cortex with different fluorescent tracers in 5 rhesus monkeys. The parietal injections included the ventral intraparietal (VIP) and lateral intraparietal (LIP) areas, and the temporal injections included the lateral portions of cytoarchitectonic areas TE and TEO. There was a remarkable segregation of cells projecting to the 2 systems. Inputs to the parietal cortex tended to arise either from areas that have been implicated in spatial or motion analysis or from peripheral field representations in the prestriate cortex. By contrast, inputs to the temporal cortex tended to arise from areas that have been implicated in form and color analysis or from central field representations. Cells projecting to the parietal cortex were found in visual area 2 (V2), but only in the far peripheral representations of both the upper and lower visual field. Likewise, labeled cells found in visual areas 3 (V3) and 4 (V4) were densest in their peripheral representations. Heavy accumulations of labeled cells were found in the dorsal parieto-occipital cortex, including the parieto-occipital (PO) area, part A of V3 (V3A), and the dorsal prelunate area (DP). In the superior temporal sulcus, cells were found within several motion-sensitive areas, including the middle temporal area (MT), the medial superior temporal area (MST), the fundus of the superior temporal area (FST), and the superior temporal polysensory area (STP), as well as within anterior portions of the sulcus whose organization is as yet poorly defined. Cells projecting to areas TE and TEO in the temporal cortex were located within cytoarchitectonic area TG at the temporal pole and cytoarchitectonic areas TF and TH on the parahippocampal gyrus, as well as in noninjected portions of area TE buried within the superior temporal sulcus. In the prestriate cortex, labeled cells were found in V2, V3, and V4, but, in contrast to the loci labeled after parietal injections, those labeled after temporal injections were concentrated in the foveal or central field representations. Although few double-labeled cells were seen, 2 regions containing intermingled parietal- and temporal-projection cells were area V4 and the cortex at the bottom of the anterior superior temporal sulcus.(ABSTRACT TRUNCATED AT 400 WORDS)

Saccadic eye movements following kainic acid lesions of the pulvinar in monkeys.

Behavioral and anatomical experiments have suggested that the pulvinar might play a role in the generation of saccadic eye movements to visual targets. To test this idea, we trained monkeys to make visually-guided saccades by requiring them to detect the dimming of a small target. We used three different saccade paradigms. On single-step trials, saccades were made from a central fixation point (FP) to a target at 12, 24 or 36 degrees to the left or right. On overlap trials, the FP remained lit during presentation of a target at 12 or 24 degrees. On double-step trials, the target stepped first to 24 degrees, and then back to 12 degrees on the same side. Animals were trained to criterion, received kainic acid lesions of the pulvinar, and were retested on all three tasks. The lesions were very large, destroying almost all of the visually responsive pulvinar. They also encroached on the lateral geniculate nucleus, thereby producing small foveal scotomas, and this resulted in some behavioral changes, including difficulty in maintaining fixation on the target and in detecting its dimming. Results on the saccade tests suggest that the pulvinar is not crucial for initiation of saccadic eye movements. Saccade latency and amplitude were unimpaired on both single-step and overlap trials. Saccadic performance was also normal on double-step trials. In a second experiment, we measured the average length of fixations during spontaneous viewing of a complex visual scene. Fixation lengths did not differ from those of unoperated control monkeys. We suggest that the neglect, increased saccadic latencies, and prolonged fixations attributed to pulvinar damage in previous studies were probably the result instead of inadvertent damage to tectal afferents. The present results, together with single unit data, point to a role for the pulvinar not in the generation of saccades, but rather in the integration of saccadic eye movements with visual processing.

Comparison of saccadic eye movements in humans and macaques to single-step and double-step target movements.

Human and monkey saccade amplitude and latency, in response to 12-36 degrees target steps, differed substantially despite nearly identical experimental conditions. On single-step trials, monkeys did not undershoot targets, and latencies were insensitive to stimulus and contextual factors. Human saccades did undershoot, their latency was longer, and both undershoot and latency were affected by stimulus variables and experimental context. On double-step trials, the second target step altered primary saccade amplitude when the step occurred as little as 40 msec prior to saccade onset for both humans and monkeys. However, humans and monkeys showed somewhat different amplitude transition functions, and monkeys showed little evidence of parallel programming.

Anterograde degeneration in the superior colliculus following kainic acid and radiofrequency lesions of the macaque pulvinar.

Several studies have reported behavioral deficits following thermocoagulation of the primate pulvinar. However, these deficits may have resulted from damage to corticotectal fibers as they pass through the pulvinar. To evaluate this possibility and to determine whether kainic acid can be used to destroy pulvinar cells without damaging corticotectal fibers, we compared anterograde degeneration in the superior colliculus following kainic acid and radiofrequency lesions of the pulvinar. Kainic acid injections into the pulvinar produced total loss of neuronal perikarya within the inferior and lateral pulvinar. Four to 7 days following the kainic acid lesions, terminal and fiber degeneration within the superior colliculus was no greater than that produced by control injections of saline. By contrast, thermocoagulation lesions of the inferior and lateral pulvinar produced dense fiber and terminal degeneration throughout the superficial and intermediate layers of the superior colliculus. We conclude that whereas thermocoagulation of the pulvinar severely damages the corticotectal tract, kainic acid lesions spare these fibers of passage. Thus kainic acid lesions should provide an effective tool for studying the functional significance of the pulvinar.

Visuotopic organization of the prelunate gyrus in rhesus monkey.

We have examined topographic organization of the prelunate gyrus and adjacent cortex buried in the lunate and superior temporal sulci. We recorded from cortex of awake rhesus monkeys performing a fixation task. Multiunit receptive fields were mapped with small, stationary spots of light to determine borders and points of strongest driving or "activity centers" of the fields. We found evidence for several distinct subdivisions of this cortex. A representation of the vertical meridian runs across the gyrus, and two crude topographic representations of the central 30 degrees of the lower quadrant, the posteromedial and anterolateral areas (area PM and area AL), share this representation of the meridian. Area AL extends from the prelunate gyrus into the posterior bank of the superior temporal sulcus; it is separated from the MT area by a narrow strip of cortex. Area PM occupies part of the prelunate gyrus and extends into the anterior bank of the lunate sulcus. Receptive field size in both AL and PM is an increasing function of eccentricity and is similar for the two areas. Medial to areas PM and AL on the prelunate gyrus is another cortical region with qualitatively different topographic organization.

Double representation of lower visual quadrant in prelunate gyrus of rhesus monkey.

The authors have mapped visuotopic organization of prelunate gyrus and adjacent buried cortex in the rhesus monkey. They found two orderly representations of the lower, contralateral visual quadrant in this cortex.

Receptive field properties of V3 neurons in monkey.

Receptive field properties of cells deep in the posterior bank of lunate sulcus and on the annectant gyrus (V3) were studied in the awake, fixating monkey. Properties of these cells were compared with those of a population of cells recorded in V2. Receptive fields of cells in V3 were larger than fields of V2 cells at comparable eccentricities. Cells were classified according to their sensitivity to the orientation and direction of motion of rectangular stimuli. Nonoriented cells were most common (39/75, 52%); the next largest class consisted of cells sensitive to stationary stimulus orientation (25/75, 33%); the third class consisted of directionally selective cells (11/75, 15%). No cells in V3 showed color-opponent responses. comparison of the orientation-sensitive population in V3 and V2 showed V3 cells more broadly tuned for orientation than V2 cells. These results suggest that V3 and V2 serve different functions in the analysis of the visual world. Neither area, however, is devoted to the analysis of only one stimulus parameter.