Craig K. Henkel

Evidence of sub-collicular auditory projections to the medial geniculate nucleus in the cat: An autoradiographic and horseradish peroxidase study

Connection 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. 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 interior 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 lemniscus and scattered cell groups around the superior olivary complex.

Development of afferent patterns in the inferior colliculus of the rat: Projection from the dorsal nucleus of the lateral lemniscus

The inferior colliculus (IC) receives a variety of layered afferent projections. The purpose of the present study was to determine the development of the projection from the dorsal nucleus of the lateral lemniscus (DNLL) to the IC in rat prior to the onset of hearing (postnatal day 12/13). Lipophilic carbocyanine dye, DiI (1,1′‐dioctodecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate), was used to trace the crossed inhibitory projection of DNLL in a developmental series of rat embryos and pups between ages embryonic day 15 (E15) and postnatal day 12 (P12). Dye‐coated pins were positioned in paraformaldehyde‐fixed brains either unilaterally in DNLL (embryonic cases), or in the commissure of Probst where DNLL fibers cross the midline (postnatal cases). By E15, pioneer fibers have left DNLL and crossed the midline. A few fibers have nearly reached the contralateral IC by E19. At birth (E22 = P0), the projection has invaded ventromedial, high‐frequency layers of the IC. The vast majority of DNLL axons parallel the presumptive IC layers by P4, and by P8 the projection has segregated into a pattern of bands (afferent dense) and interband (afferent sparse) spaces that encompasses the entire frequency axis of the IC. Adult‐like patches, regions along afferent bands that exhibit the heaviest labeling, develop by P12. These results indicate that some mature projection patterns are in place prior to the onset of hearing. Such findings suggest that evoked activity may not be required for the initial organization of patterned projections in the ascending auditory pathway. J. Comp. Neurol. 416:368‐382, 2000.

Localization of the motor neurons to the tensor tympani muscle.

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.

Quantitative Changes in Calretinin Immunostaining in the Cochlear Nuclei after Unilateral Cochlear Removal in Young Ferrets

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. J. Comp. Neurol. 483:458–475, 2005.

Alterations in calretinin immunostaining in the ferret superior olivary complex after cochlear ablation

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.

Calcium-binding proteins and GABA reveal spatial segregation of cell types within the developing lateral superior olivary nucleus of the ferret

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. Microsc. Res. Tech. 41:234–245, 1998.

Evidence of collateral axonal projections to the superior olivary complex

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.

Cochlear ablation in adult ferrets results in changes in insulin-like growth factor-1 and synaptophysin immunostaining in the cochlear nucleus

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.

Upregulation of calretinin immunostaining in the ferret inferior colliculus after cochlear ablation

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.

Anterior hippocampal volume is reduced in behaviorally depressed female cynomolgus macaques

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 10th section throughout the extent of the HC was manually traced and used to reconstruct the 3D 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.