Search

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.

Converging lines of behavioral and pharmacological evidence suggest that GABAergic synapses in the basolateral amygdala (BLA) may play an integral role in mediating the anxiolytic effects of ethanol (EtOH). Since anxiety is thought to play an important role in the development of, and relapse to, alcoholism, elucidating the mechanisms through which EtOH modulates GABAergic synaptic transmission in the BLA may be fundamental in understanding the etiology of this disease. A recent study in mice has shown that principal cells within the BLA receive inhibitory input from two distinct types of GABAergic interneurons: a loosely distributed population of local interneurons and a dense network of paracapsular (pcs) GABAergic cells clustered along the external capsule border. Here, we sought to confirm the presence of these two populations of GABAergic synapses in the rat BLA and evaluate their ethanol sensitivity. Our results suggest that rat BLA pyramidal cells receive distinct inhibitory input from local and pcs interneurons and that EtOH potentiates both populations of synapses, albeit via distinct mechanisms. EtOH enhancement of local inhibitory postsynaptic currents (IPSCs) was associated with a significant decrease in paired-pulse ratio (PPR) and was significantly potentiated by the GABA(B) receptor antagonist SCH 50911 [(+)-(S)-5,5-dimethylmorpholinyl-2-acetic acid], consistent with a facilitation of GABA release from presynaptic terminals. Conversely, EtOH enhancement of pcs IPSCs did not alter PPR and was not enhanced by SCH 50911 but was inhibited by blockade of noradrenergic receptors. Collectively, these data reveal that EtOH can potentiate GABAergic inhibitory synaptic transmission in the rat BLA through at least two distinct pathways.

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 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.

Four old (29 months) Brown Norway×Fischer 344 (BN×F344) rats received intracerebroventricular infusion of insulin-like growth factor-1 (IGF-1) and four middle-aged (18 months) and four old (29 months) rats received infusion of saline for 28 days. Sensorimotor cortex containing Layer 2 was blocked and processed for electron microscopy. Thin (700 A) and semithin (1 μm) sections were collected from the same anatomical space for quantification of synapses and neurons, respectively, using the physical disector. Numerical density (Nv) of presumptive inhibitory synapses in Layer 2 of sensorimotor cortex has been reported to decline with age Poe et al., 2001, Brunso-Bechtold et al. [Brain Res. 872 (2000) 125]. Infusion of IGF-1 did not affect the density of synapses or neurons when old IGF-1 animals were compared with old saline animals.

Organotypic slices are used frequently in studies of central nervous system development and function because they provide excellent experimental access with significant preservation of cellular context and relationships. Within a slice, however, a variety of factors may cause individual classes of neurons to respond differently to the culture environment. Differences in deafferentation, cellular maturation, trophic dependence and ongoing naturally occurring cell death may produce changes in the neuronal population that are transparent to the experimenter but that could affect experimental results significantly. In this study, we examined the distribution and prevalence of cell death among neurons in each cortical layer in organotypic slices. In addition, we assessed the ability of several neurotrophic factors to ameliorate neuronal death in each cortical layer. Within the first 24 hr in culture, there was striking laminar variation in the extent of neuronal death in culture, which could not be accounted for by the pattern of programmed cell death in vivo. In addition, neurons in the six layers of the neocortex differed in the degree to which they could be rescued by neurotrophic factors. These data suggest that differential neuronal death and rescue are important considerations in studies utilizing organotypic slices and may represent particularly confounding variables in studies of effects of trophic factors in such preparations. J. Neurosci. Res. 65:455–462, 2001. © 2001 Wiley-Liss, Inc.

The synapse, as the site of functional neural interaction, has been suggested as a possible substrate for age-related impairment of cognitive ability. Using the physical disector probe with tissue prepared for ultrastructural analysis, we find an age-related decline in the numerical density of presumptive inhibitory synapses in layer 2 of the sensorimotor cortex of the Brown Norway × Fisher 344 rat. This age-related decline in presumptive inhibitory synapses is maintained when the density of synapses is combined with the numerical density of neurons quantified from the same anatomical space to arrive at a ratio of synapses per neuron. The numerical density of these synapses declines between middle-aged (18 months) and old (29 months) animals by 36% whereas numerical density of neurons does not change between these ages, resulting in a decline in the ratio of presumptive inhibitory synapses per neuron in this cortical area. This study demonstrates a deficit in the intrinsic inhibitory circuitry of the aging neocortex, which suggests an anatomical substrate for age-related cognitive impairment. J. Comp. Neurol. 439:65–72, 2001. © 2001 Wiley-Liss, Inc.

Investigations of age-related changes in synapse density have yielded contradictory conclusions. The goal of the present study was to determine whether there is a significant decline in the number of cortical synapses in old age. Therefore, brains from 10-, 15-, and 32-month-old Brown Norway X Fischer 344 rats were prepared for electron microscopy and synapses were counted in a stereotaxically-identified region of sensorimotor cortex. Within this cortical area, synapses were counted in layers 2 and 4 and the data have been presented both as number of synapses per volume of neuropil and as the ratio of synapses per neuron. Results indicated that there was a decline in synapse density between 15 and 32 months in layer 2, but not in layer 4. This decline was significant not only for total synapses, but also for subcategories of synapses when classified by presynaptic features or postsynaptic element. Specifically, there was a significant decline in presumptive inhibitory synaptic terminals, i.e., those containing nonround synaptic vesicles, as well as a significant decline in synapses that contact dendritic spines.

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.

In order to study the initial outgrowth of cochlear nucleus fibers, the lipophilic carbocyanine dye DiI was used to label these neurites in rats at E13 and E15. At E13, prior to leaving the ventricular zone, cochlear neurons have already extended axons along the marginal edge of the hindbrain and a few fibers have reached the midline. By E15, many more fibers have entered the midline region and some appear to contact glial cells at the midline. Based on axon trajectory and growth cone morphology it does not appear likely that there are significant intermediate decision points for trapezoid body fibers at these early stages of development. Contact between growth cones and glial cells at the midline is consistent with these cells playing a role in providing guidance cues for developing auditory fibers.

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.

We examined cultures of purified type 1 astrocytes and mixed glial co-cultures containing type 1 astrocytes and 0-1A lineage cells in media containing fetal calf serum at 5 days in vitro (DIV), 12 DIV, and 30 DIV, using cell-specific immunocytochemical markers and electron microscopy. At all three time points and in both culture systems, the polygonal-shaped type 1 astrocytes were A2B5−, GFAP+, and GalC− (specific markers for 0-2A lineage cells, and mature astrocytes and oligodendrocytes, respectively). From 5 to 30 DIV, the type 1 astrocytes increased markedly in size and the appearance of the cytoskeleton changed dramatically, with the amount of glial filaments increasing and microtubules decreasing. At 5, 12, and 30 DIV, the 0-2A lineage cells were multipolar, A2B5+, HNK-1+, GFAP−, and GalC−. The 0-2A lineage cells could not be distinguished as either astrocytes or oligodendrocytes on the basis of immunocytochemical or ultrastructural characteristics. These cells had dense cytoplasm, very few intermediate filaments, and a large number of vacuoles and dense bodies. The general characteristics of the cultured astrocytes at 12 DIV and 30 DIV were similar to mature and aged astrocytes in vivo, respectively. These findings suggest that the culture environment in this study accelerated aging of type 1 astrocytes. 0-2A lineage cells, on the other hand, appeared unable to differentiate into either type 2 astrocytes or oligodendrocytes when cultured in the presence of both type 1 astrocytes and fetal calf serum.

Greater than 50% of adults and ~ 100% of children who survive > 6 months after fractionated partial or whole-brain radiotherapy develop cognitive impairments. Noninvasive methods are needed for detecting and tracking the radiation-induced brain injury associated with these impairments. Using magnetic resonance imaging, we sought to detect structural changes associated with brain injury in our rodent model of fractionated whole-brain irradiation (fWBI) induced cognitive impairment and to compare those changes with alterations that occur during the aging process. Middle aged rats were given a clinically relevant dose of fWBI (40 Gy: two 5 Gy fractions/week for 4 weeks) and scanned approximately 1 year post-irradiation to obtain whole-brain T2 and diffusion tensor images (DTI); control groups of sham-irradiated age-matched and young rats were also scanned. No gross structural changes were evident in the T2 structural images, and no detectable fWBI-induced DTI changes in fractional anisotropy (FA) were found in heavily myelinated white matter (corpus callosum, cingulum, and deep cortical white matter). However, significant fWBI-induced variability in FA distribution was present in the superficial parietal cortex due to an fWBI-induced decline in FA in the more anterior slices through parietal cortex. Young rats had significantly lower FA values relative to both groups of older rats, but only within the corpus callosum. These findings suggest that targets of the fWBI-induced change in this model may be the less myelinated or unmyelinated axons, extracellular matrix, or synaptic fields rather than heavily myelinated tracts.

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.

Radiation therapy is used widely to treat primary and metastatic brain tumors, but also can lead to delayed neurological complications. Since maintenance of myelin integrity is important for cognitive function, the present study used a rat model that demonstrates spatial learning and memory impairment 12 months following fractionated whole-brain irradiation (WBI) at middle age to investigate WBI-induced myelin changes. In this model, 12-month Fischer 344 x Brown Norway rats received 9 fractions of 5 Gy delivered over 4.5 weeks (WBI rats); Sham-IR rats received anesthesia only. Twelve months later, the brains were collected and measures of white matter integrity were quantified. Qualitative observation did not reveal white matter necrosis one year post-WBI. In addition, the size of major forebrain commissures, the number of oligodendrocytes, the size and number of myelinated axons, and the thickness of myelin sheaths did not differ between the two groups. In summary, both the gross morphology and the structural integrity of myelin were preserved one year following fractionated WBI in a rodent model of radiation-induced cognitive impairment. Imaging studies with advanced techniques including diffusion tensor imaging may be required to elucidate the neurobiological changes associated with the cognitive impairment in this model.

Purpose To determine whether hippocampal neurons are lost 12 months after middle-aged rats received a fractionated course of whole-brain irradiation (WBI) that is expected to be biologically equivalent to the regimens used clinically in the treatment of brain tumors. Methods and Materials Twelve-month-old Fischer 344 X Brown Norway male rats were divided into WBI and control (CON) groups (n = 6 per group). Anesthetized WBI rats received 45 Gy of 137Cs γ rays delivered as 9 5-Gy fractions twice per week for 4.5 weeks. Control rats were anesthetized but not irradiated. Twelve months after WBI completion, all rats were anesthetized and perfused with paraformaldehyde, and hippocampal sections were immunostained with the neuron-specific antibody NeuN. Using unbiased stereology, total neuron number and the volume of the neuronal and neuropil layers were determined in the dentate gyrus, CA3, and CA1 subregions of hippocampus. Results No differences in tissue integrity or neuron distribution were observed between the WBI and CON groups. Moreover, quantitative analysis demonstrated that neither total neuron number nor the volume of neuronal or neuropil layers differed between the two groups for any subregion. Conclusions Impairment on a hippocampal-dependent learning and memory test occurs 1 year after fractionated WBI at middle age. The same WBI regimen, however, does not lead to a loss of neurons or a reduction in the volume of hippocampus.

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.

Caloric restriction (CR) can attenuate the aging-related decline in learning and memory in rats. Understanding the mechanisms underlying this effect could lead to therapies for human memory impairment. We tested the hypotheses that aging is associated with a decline in hippocampal brain-derived neurotrophic factor (BDNF), a growth factor that enhances learning and memory, and that CR increases hippocampal BDNF. We compared BDNF protein levels in hippocampal subregions of young, middle-aged and old rats fed CR or ad libitum (AL) diets. Mean BDNF levels in the dentate gyrus and CA3 did not differ with diet but increased with age. In CA1, BDNF levels were slightly higher in CR than AL rats at middle and old age but did not change across lifespan. These data suggest that mnemonic impairments with age do not reflect a decrease in hippocampal BDNF. Furthermore, if CRs attenuation of aging-related memory changes is mediated by BDNF, then it must be through a small, CA1-specific increase and does not involve reversal of an aging-related decline in BDNF.

Previous studies have demonstrated an age-related decline in the density of presumptive inhibitory synapses in layer 2 of rat sensorimotor cortex [J. Comp. Neurol. 439(1) (2001) 65]. Caloric restriction has been shown to ameliorate age-related deterioration in a variety of systems and to extend life span. The present study tested the hypothesis that caloric restriction would prevent the previously reported age-related synaptic decline. Accordingly, synaptic density in layer 2 of sensorimotor cortex was compared between 29-month-old male rats fed ad libitum and 29-month-old male rats that were caloric restricted (60% of ad libitum calories) from 4 months of age. In serial electron micrographs, the physical disector was used to determine the numerical density of presumptive excitatory and inhibitory synapses (those containing round or nonround vesicles, respectively) as well as that of neurons. Not only was the previously reported age-related decline in numerical density of presumptive inhibitory synapses not ameliorated by caloric restriction, the numerical density was significantly lower in caloric restricted than in ad libitum fed rats for total as well as for presumptive excitatory and inhibitory synapses. Analysis further revealed no difference in the numerical density of neurons in this region. Relating synapse density to neuron density as the ratio of synapses to neuron also demonstrated significantly fewer synapses per neuron in caloric restricted than in ad libitum fed old rats. Finally, synapse length was significantly less in caloric restricted rats. These results suggest that not only does caloric restriction fail to prevent the age-related decline in presumptive inhibitory synapses, it results in fewer presumptive excitatory synapses as well.

Age-related effects associated with the hippocampus include declines in numbers of neurons and synapses in the dentate gyrus and area CA1, and decreased cognitive ability as assessed with the Morris water maze. The present study quantified both neuron and synapse number in the same tissue block of area CA3 of the hippocampus. No investigations of both density of neurons and synapses together in area CA3 of hippocampus have been performed previously, despite its importance as the terminal field of dentate gyrus mossy fibers, the second synapse in the trisynaptic circuit in the hippocampus. Numerical density of neurons and synapses were assessed in 4-, 18-, and 29-month-old rats receiving infusions of saline into the lateral ventricle and in 29-month-old rats receiving infusions of insulin-like growth factor-1 (IGF-1). Numerical density of neurons of the stratum pyramidale of CA3 of hippocampus remained constant across the life span as did the numerical density of synapses in stratum lucidum of area CA3. Despite the reported role of IGF-1 in synaptogenesis and improvements in behavior with age, ventricular infusion of this growth factor did not affect the numerical density of neurons or synapses in 29-month-old rats when compared to saline-infused old rats. Further, reported effects of IGF-1 on adult neurogenesis in the dentate gyrus are not reflected in an IGF-1-related increase in synapse density in this region.

The temporal and spatial distributions of several growth factors suggest roles in the regulation of neuronal differentiation in the neocortex. Among such growth factors, the insulin-like growth factors (IGF-I and -II) are of particular interest because they are available to neurons from multiple sources under independent control. IGF-I is produced by many neurons throughout the brain and also by cells in the cerebral vasculature. IGF-II is found at high levels in the CSF, and both IGF-I and IGF-II cross the blood–brain barrier. Thus, the IGFs may act as both paracrine and endocrine regulators of neuronal development. As an initial step toward understanding the influence of IGFs in the developing cerebral cortex, the present study examined the effects of IGF-I and of the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) on the dendritic complexity of layer 2 pyramidal neurons. The results demonstrate that IGF-I increased the branching and total extent of both apical and basal dendrites of pyramidal cells in organotypic slices of rat primary somatosensory cortex. BDNF and NT-3 also enhanced dendritic development, but the two neurotrophins increased the extent of only basal, not apical, dendrites and promoted greater elongation than was seen after IGF-I treatment. These results provide direct evidence that IGF-I can regulate the dendritic elaboration of cortical neurons and indicate that endogenous IGFs may influence dendritic differentiation and the formation of cortical connections. In addition, IGF-dependent regulation of dendritic structure may represent a link between age-related declines in IGFs and cognitive deficits seen in senescence.

N-Methyl-D-aspartate (NMDA) receptors have been reported to have an important role in synaptic plasticity and neurodegeneration. Two major subtypes of these receptors, NMDAR1 and NMDAR2, are present in brain and heterogeneity of these receptors have been reported to define specific functional responses. In this study, the effects of age and chronic insulin-like growth factor-1 (IGF-1) administration on NMDA receptor density and subtype expression were investigated in frontal cortex, CA1, CA2/3 and the dentate gyrus of the hippocampus of young (10 months), middle-aged (21 months) and old (30 months) male Fisher 344xBrown Norway (F1) rats. No age-related changes in (125)I-MK-801 binding or NMDAR1 protein expression were observed in hippocampus or frontal cortex. However, analysis of NMDAR2A and NMDAR2B protein expression in hippocampus indicated a significant decrease between 21 and 30 months of age and administration of IGF-1 increased these receptor subtypes. In cortex, NMDAR2A and NMDAR2B protein expression were not influenced by age or IGF-1 treatment, although NMDAR2C protein expression decreased with age and this decline was not ameliorated by IGF-1 administration. These data demonstrate that NMDA receptor subtypes are altered with age in a regional and subtype specific manner. We conclude that both age and IGF-1 regulate the expression of NMDA receptor subtypes and suggest that age-related changes in NMDA receptor heterogeneity may result in functional changes in the receptor that have relevance for aging.

Previous studies have reported changes in insulin-like growth factor I (IGF-I) mRNA expression during early postnatal development of the rat brain. Although changes in IGF-I gene expression have been documented in a wide range of central nervous system structures during early development and investigated in the hippocampus during aging, no study has compared changes in IGF-I gene expression in different brain regions across the life span. The present study assessed the distribution of IGF-I gene expression using in situ hybridization in rats aged 2-30 months. Dot blots were used as a quantitative assessment of cortical IGF-I mRNA. Results indicate that both the distribution and levels of brain IGF-I mRNA do not change significantly between 2 and 30 months of age in the rat. However, in spite of relatively constant levels of mRNA, other studies from our laboratory have demonstrated that cortical IGF-I protein levels decrease 36.6% between 11 and 32 months of age, suggesting that IGF-I function is decreased with increasing age.

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 present study examined the fate and migration of transplanted astrocytes in different host ages. Additionally, the effect of donor cell age was examined in relation to cell migration. Cultured astrocytes from 5,12 and 30 days in vitro were transplanted into young (postnatal day 5 and 21), adult (4.5 month), and aged (21 month) animals. The transplanted cells were labeled with Fast Blue, Fluorogold or DiI. The results confirmed previous studies demonstrating that transplanted cells were able to migrate successfully through host central nervous system and extended those findings to show that the age of the host significantly influenced donor cell migration distance. Migration was most extensive in young animals, as conditions supporting cell migration appeared to be lacking in older animals. Donor cells preferentially migrated on myelinated fiber tracts, rather than on unmyelinated fiber tracts or gray matter. The donor cells were not glial fibrillary acidic protein positive, indicating that either the cultured type 1 astrocytes did not survive transplantation or underwent significant remodeling of the intermediate filament network. It is also possible that a subpopulation of cells, possibly immature astrocytes which are present in the transplanted cell suspensions, flourished and subsequently migrated in the host brains.

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.

In recent years, the important role of the extracellular matrix in neural development has been increasingly recognized. In order to begin to examine what role might be played by the extracellular matrix in the developing dorsal lateral geniculate nucleus (dLGN), the present study used immunocytochemistry to assess the distribution of a recognized extracellular matrix molecule, fibronectin (FN), during postnatal development of the ferret dLGN. Prior to the segregation of cell layers, no clear pattern of FN distribution can be distinguished within the dLGN. By P16, when layers A and A1 are separated by an interlaminar space, FN is localized in that space. By P24, FN is present not only between layers A and A1, but also within layers A and A1 as bands parallel to the laminar borders. These bands appear to correspond spatially and temporally to the development of sublaminar boundaries in the ferret (Hahm and Sur, Neurosci Abstr 14:460, 1988). By the end of the first postnatal month, immunoreactivity is diminished but still present. FN is no longer present at P44 or in the adult. Adjacent sections were incubated with antiserum to glial fibrillary acid protein (GFAP). When a laminar pattern of GFAP can be distinguished, it coincides with the interlaminar distribution of FN. The findings described here are consistent with a role for FN in the development of a laminar organization in the dLGN.

The development of neurons in the dorsal lateral geniculate nucleus (dLGN) of pigmented ferrets was studied by using the Golgi-Hortega technique. In adult ferrets, four dLGN cell classes were defined on the basis of somatic and dendritic morphology. Classes 1 and 2 were divided into stellate and oriented subtypes. Class 1 and 4 cells are characterized by filiform appendages, class 2 cells by club-like appendages, and class 3 cells by stalked appendages. At birth, dLGN neurons have simple dendritic arbors. During the first postnatal week, dendritic length and proximal branching density increase markedly. By postnatal day 21 (P21), dendritic morphology begins to take on mature characteristics and by the time of eye opening (P30-P35), most neurons can be classified. Also by that time, dLGN cells are covered with abundant filiform appendages. Developmental changes in appendage density were quantified for class 1 stellate cells. These data reveal that appendage density reaches a peak at P56, decreases sharply until P90, and then gradually declines to mature levels by P180. Elaboration and elimination of transient appendages occurs centrifugally; at maturity appendage density remains greater distally.

In normal development, cell layers in the dorsal lateral geniculate nucleus (dLGN) segregate from a relatively homogeneous cell group. If all retinal input is removed prior to this segregation, the layers fail to form. In the present study, we used ultrastructural and morphometric analyses to study dLGN development in the tree shrew following neonatal removal of retinal input. The goal of the present study was to determine whether there are differences between normal animals and enucleates in the development of dLGN cells and their interrelationships with each other and/or with the surrounding glia, which might explain the failure of cellular lamination in enucleated animals. The results indicate that although the development in enucleated animals may take place somewhat more slowly, by P90 cell size and density are not significantly different from normal. These results, coupled with the observation that the dLGN in enucleates is smaller than in normals, suggest that the removal of retinal input results in dLGN cell loss. At both the light and electron microscopic level, cells in the developing normal dLGN are arranged in bands of immediately adjacent cells. In enucleates, dLGN cells are less frequently in immediate contact and are arranged in small groups or clumps which may be separated by degenerating cells. The present data suggest that the presence of retinal input may be necessary to allow dLGN cells to maintain the intercellular relationships necessary for laminar segregation to take place.

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.

In the spinal ganglia of the chick embryo, two neuronal populations can be distinguished: large, early differentiating ventrolateral (VL) cells and small, late differentiating dorsomedial (DM) cells. It was found that, beginning with stage 25, the DM cells originate from a narrow band of small, immature cells at the medial border of the ganglion, extending to the dorsolateral border. We have designated this band as the inner and outer marginal zone. Neuronal death was investigated in thoracic ganglion 18 and brachial ganglion 15 by counting degenerating cells, separately for the VL and DM populations, at every stage from stage 24 (4% days) to stage 38 (12 days). In both ganglia, separate degeneration periods were found for the VL and DM populations which do not overlap. The peaks of degeneration are: stage 27 (5% days) for the VL population in ganglion 18, stage 30 (6% to 7 days) for VL in ganglion 16, and stage 35 (8% days) for DM in both ganglia. Daily injections of 6 pg of nerve growth factor, (NGF) in 6 to 12 ~1 of 0.9% sterile salt solution into the yolk sac from stage 21 (3% days) to the day of sacrifice resulted in a significant reduction of neuronal death in the VL population of ganglion 18 and in the rescue of practically all VL neurons in ganglion 15 and all DM neurons in both ganglia, which normally would have died. This is the first demonstration of an NGF effect on VL neurons. In the analysis of the multiple effects of nerve growth factor (NGF), the sympathetic ganglia have played the dominant role; the sensory ganglia which are the other target of NGF have attracted much less attention. Yet, if one entertains the notion that NGF might be the natural trophic maintenance agent for these two neuron types (i.e., that NGF is actually produced by their target organs), then one approach to the testing of this hypothesis would be to engage in in viva experiments. For this

In the present study, we examined the colliculogeniculate projection in normal adult tree shrews and in adults that were bilaterally enucleated at birth. We injected lectin-conjugated HRP into superficial superior colliculus and then mapped the pattern of anterogradely transported enzyme in the ipsilateral dLGN. In normal adult tree shrews, the results confirm that the colliculogeniculate projection is laminated and terminates predominantly in small-celled layers 3 and 6 and in the interlaminar space between layers 4 and 5 (Fitzpatrick et al., 1980); we report an additional sparse projection to layer 4. In bilaterally enucleated animals, the colliculogeniculate projection is unlaminated and tends to terminate in the lateral two-thirds of the dLGN even though synaptic sites are vacated throughout the nucleus. We suggest that this preference may be due to a specificity of the colliculogeniculate fibers for the lateral two-thirds of the dLGN, which, in normal adult tree shrews, contains cells with similar physiological characteristics. We further suggest that the normal lamination of the colliculogeniculate projection in the lateral two- thirds of the dLGN may be due to competition with retinogeniculate fibers so that colliculogeniculate fibers terminate predominantly in layers containing small, W-like cells.

The role of retinogeniculate afferents in the development of patterns of connections between visual cortex and the lateral geniculate nucleus (LGN) was addressed by studying the effect of bilateral enucleation at birth on those patterns of connections in tree shrew. In normal adult tree shrews there are six LGN cell layers separated by cell-sparse interlaminar spaces. The reciprocal connections between the LGN and visual cortex are restricted to a column running across all six LGN layers; the geniculocortical projection arises from the cell layers while the corticogeniculate projection terminates primarily in the interlaminar spaces. At birth, when the experimental animals were bilaterally enucleated, the retinogeniculate fibers have begun to segregate by eye but neither the cytological characteristics of individual layers nor the interlaminar spaces have yet formed, and the corticogeniculate fibers have not entered the nucleus. Bilateral enucleation does not prevent the development of the cytological characteristics of individual layers but the interlaminar spaces do not develop. The results of [3H]proline/HRP injections into visual cortex in animals bilaterally enucleated at birth indicate that in the absence of retinogeniculate fibers, and thus interlaminar spaces, the corticogeniculate fibers do not concentrate at the laminar borders but instead spread across all six LGN cell layers. Despite the failure of this projection to concentrate at laminar borders, the corticogeniculate fibers do terminate within a restricted projection column.

In the present study, we quantified the distribution of growth cones and synapses in 2 developing layers, as well as in the intervening interlaminar space of the dorsal lateral geniculate nucleus (dLGN) in tree shrews. Our goal was to gain insight into mechanisms involved in the segregation of dLGN cells into layers during development. We sacrificed tree shrews before (P0), during (P4 and P7), and after (P15) laminar segregation as well as at maturity (P90). The dLGN from each animal was sectioned horizontally, and all tissue for analysis was blocked from the middle third of the nucleus along the dorsoventral axis. Each micrograph was coded and blindly scored for the number of growth cones and synapses in layers 4 and 5 and the intervening interlaminar space. We also measured each growth cone and classified synapse type. Statistical analyses of these data reveal that neither growth cones nor synapses are significantly more common in the interlaminar space early in the period of laminar segregation (P4). By nearly a week after the interlaminar space can first be distinguished (P7), there are more growth cones in the interlaminar space than in the layers, but this difference is no longer present at (P15). These results suggest that, although neuropil development at the laminar borders may not play a role in the onset of laminar segregation, it may contribute to the widening of the interlaminar spaces once this process has begun. In addition, growth cones continuously decrease in number and become less bulbous and more linear in shape with development. Synapses, on the other hand, continuously increase in number with age and pass through a transient period characterized by heavy spinous terminations.

In order to gain insight into the mechanisms involved in the formation of groupings of functionally similar cells in the developing nervous system, we have studied the formation of cell layers in the developing dorsal lateral geniculate nucleus (dLGN). To examine the possibility that a higher affinity or adhesion between cells in individual layers may play a role in laminar segregation, we studied cellular interrelationships in the dLGN of tree shrews before (P0), during (P4 and P7), and just after (P15) laminar segregation has taken place. We compared our observations at these stages of development with similar observations in the adult. In none of the cases do we see evidence of gap junctions either between adjacent neurons or between neurons and processes in the surrounding neuropil. However, we frequently observe the presence of puncta adherentes between adjacent neurons at all stages of development. These profiles are also present between neurons and cellular processes in the neuropil. We also see subsurface cisternae in all of our cases, although these are more pronounced before and during interlaminar space formation. As with the puncta adherentes, these are found both between adjacent neurons as well as between neurons and other elements in the neuropil. We also see some evidence of what appear to be cytoplasmic bridges between adjacent neurons; these are quite rare but appear to be present only before and during laminar segregation. Finally, we frequently see cytoplasmic processes interdigitated between otherwise immediately adjacent cells. These processes also are often found oriented along other portions of the neuronal plasmalemma. Whether these processes are portions of neuronal growth cones or glial processes is impossible to determine at this time. Because of the potential role glial processes may play in the formation and maintenance of laminar cell groupings during layer formation, we have also made a preliminary survey of whether glial cells can be distinguished ultrastructurally at the stages we have studied.