Growth hormone and insulin-like growth factor-I alter hippocampal excitatory synaptic transmission in young and old rats.

In rats, as in humans, normal aging is characterized by a decline in hippocampal-dependent learning and memory, as well as in glutamatergic function. Both growth hormone (GH) and insulin-like growth factor-I (IGF-I) levels have been reported to decrease with age, and treatment with either GH or IGF-I can ameliorate age-related cognitive decline. Interestingly, acute GH and IGF-I treatments enhance glutamatergic synaptic transmission in the rat hippocampus of juvenile animals. However, whether this enhancement also occurs in old rats, when cognitive impairment is ameliorated by GH and IGF-I (des-IGF-I), remains to be determined. To address this issue, we used an in vitro CA1 hippocampal slice preparation and extracellular recording techniques to study the effects of acute application of GH and IGF-I on compound field excitatory postsynaptic potentials (fEPSPs), as well as AMPA- and NMDA-dependent fEPSPs, in young adult (10 months) and old (28 months) rats. The results indicated that both GH and IGF-I increased compound-, AMPA-, and NMDA-dependent fEPSPs to a similar extent in slices from both age groups and that this augmentation was likely mediated via a postsynaptic mechanism. Initial characterization of the signaling cascades underlying these effects revealed that the GH-induced enhancement was not mediated by the JAK2 signaling element in either young adult or old rats but that the IGF-I-induced enhancement involved a PI3K-mediated mechanism in old, but not young adults. The present findings are consistent with a role for a GH- or IGF-I-induced enhancement of glutamatergic transmission in mitigating age-related cognitive impairment in old rats.

Imaging radiation-induced normal tissue injury.

Technological developments in radiation therapy and other cancer therapies have led to a progressive increase in five-year survival rates over the last few decades. Although acute effects have been largely minimized by both technical advances and medical interventions, late effects remain a concern. Indeed, the need to identify those individuals who will develop radiation-induced late effects, and to develop interventions to prevent or ameliorate these late effects is a critical area of radiobiology research. In the last two decades, preclinical studies have clearly established that late radiation injury can be prevented/ameliorated by pharmacological therapies aimed at modulating the cascade of events leading to the clinical expression of radiation-induced late effects. These insights have been accompanied by significant technological advances in imaging that are moving radiation oncology and normal tissue radiobiology from disciplines driven by anatomy and macrostructure to ones in which important quantitative functional, microstructural, and metabolic data can be noninvasively and serially determined. In the current article, we review use of positron emission tomography (PET), single photon emission tomography (SPECT), magnetic resonance (MR) imaging and MR spectroscopy to generate pathophysiological and functional data in the central nervous system, lung, and heart that offer the promise of, (1) identifying individuals who are at risk of developing radiation-induced late effects, and (2) monitoring the efficacy of interventions to prevent/ameliorate them.

Growth hormone modulates hippocampal excitatory synaptic transmission and plasticity in old rats.

Alterations in the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPA-R) and N-methyl-D-aspartate receptor (NMDA-R) have been documented in aged animals and may contribute to changes in hippocampal-dependent memory. Growth hormone (GH) regulates AMPA-R and NMDA-R-dependent excitatory transmission and decreases with age. Chronic GH treatment mitigates age-related cognitive decline. An in vitro CA1 hippocampal slice preparation was used to compare hippocampal excitatory transmission and plasticity in old animals treated for 6-8 months with either saline or GH. Our findings indicate that GH treatment restores NMDA-R-dependent basal synaptic transmission in old rats to young adult levels and enhances both AMPA-R-dependent basal synaptic transmission and long-term potentiation. These alterations in synaptic function occurred in the absence of changes in presynaptic function, as measured by paired-pulse ratios, the total protein levels of AMPA-R and NMDA-R subunits or in plasma or hippocampal levels of insulin-like growth factor-I. These data suggest a direct role for GH in altering age-related changes in excitatory transmission and provide a possible cellular mechanism through which GH changes the course of cognitive decline.

Aging masks detection of radiation-induced brain injury.

Fractionated partial or whole-brain irradiation (fWBI) is a widely used, effective treatment for primary and metastatic brain tumors, but it also produces radiation-induced brain injury, including cognitive impairment. Radiation-induced neural changes are particularly problematic for elderly brain tumor survivors who also experience age-dependent cognitive impairment. Accordingly, we investigated i] radiation-induced cognitive impairment, and ii] potential biomarkers of radiation-induced brain injury in a rat model of aging. Fischer 344 x Brown Norway rats received fractionated whole-brain irradiation (fWBI rats, 40 Gy, 8 fractions over 4 weeks) or sham-irradiation (Sham-IR rats) at 12 months of age; all analyses were performed at 26-30 months of age. Spatial learning and memory were measured using the Morris water maze (MWM), hippocampal metabolites were measured using proton magnetic resonance spectroscopy ((1)H MRS), and hippocampal glutamate receptor subunits were evaluated using Western blots. Young rats (7-10 months old) were included to control for age effects. The results revealed that both Sham-IR and fWBI rats exhibited age-dependent impairments in MWM performance; fWBI induced additional impairments in the reversal MWM. (1)H MRS revealed age-dependent decreases in neuronal markers, increases in glial markers, but no detectable fWBI-dependent changes. Western blot analysis revealed age-dependent, but not fWBI-dependent, glutamate subunit declines. Although previous studies demonstrated fWBI-induced changes in cognition, glutamate subunits, and brain metabolites in younger rats, age-dependent changes in these parameters appear to mask their detection in old rats, a phenomenon also likely to occur in elderly fWBI patients >70 years of age.

Differential effects of radiation and age on diffusion tensor imaging in rats.

Greater than 50% of adults and approximately 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.

Maintenance of white matter integrity in a rat model of radiation-induced cognitive impairment.

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.

Stability of local brain levels of insulin-like growth factor-I in two well-characterized models of decreased plasma IGF-I.

Insulin-like growth factor-I (IGF-I), a functionally important neurotrophic factor, impacts tissues throughout the body including the central nervous system. In addition to the significant proportion of IGF-I that is synthesized in the liver and released into the plasma, IGF-I is expressed locally in tissues. The present study investigated the relationship between plasma and local brain levels of IGF-I in two well-characterized models of decreased IGF-I. The first is an adult-onset growth hormone deficiency (AOGHD) model, and the second is a caloric restriction (CR) model. In the first cohort of animals from both models, the hippocampus was removed from the brain immediately following decapitation, and in the second cohort, the animals were perfused transcardially with phosphate buffered saline to remove cerebral blood prior to harvesting the hippocampus. Our results demonstrated that although the plasma IGF-I levels were decreased in the CR and AOGHD rats compared to controls, the hippocampal IGF-I levels did not differ among the groups. These data suggest that local brain IGF-I levels are regulated in a different manner than plasma IGF-I levels.

Hippocampal neuron number is unchanged 1 year after fractionated whole-brain irradiation at middle age.

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 (137)Cs gamma 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.

Caloric restriction and age affect synaptic proteins in hippocampal CA3 and spatial learning ability.

Caloric restriction (CR) is a daily reduction of total caloric intake without a decrease in micronutrients or disproportionate reduction of any one dietary component. CR can increase lifespan reliably in a wide range of species and appears to counteract some aspects of the aging process throughout the body. The effects on the brain are less clear, but moderate CR seems to attenuate age-related cognitive decline. Thus, we determined the effects of age and CR on key synaptic proteins in the CA3 region of the hippocampus and whether these changes were correlated with differences in behavior on a hippocampal-dependent learning and memory task. We observed an overall, age-related decline in the NR1, N2A and N2B subunits of the N-methyl-d-aspartate (NMDA)-type and the GluR1 and GluR2 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)-type ionotropic glutamate receptors. Interestingly, we found that CR initially lowers the glutamate receptor subunit levels as compared to young AL animals, and then stabilizes the levels across lifespan. Synaptophysin, a presynaptic vesicle protein, showed a similar pattern. We also found that both CR and ad libitum (AL) fed animals exhibited age-related cognitive decline on the Morris water maze task. However, AL animals declined between young and middle age, and between middle age and old, whereas CR rats only declined between young and middle age. Thus, the decrease in key synaptic proteins in CA3 and cognitive decline occurring across lifespan are stabilized by CR. This age-related decrease and CR-induced stabilization are likely to affect CA3 synaptic plasticity and, as a result, hippocampal function.

Effects of aging and caloric restriction on dentate gyrus synapses and glutamate receptor subunits.

Caloric restriction (CR) attenuates aging-related degenerative processes throughout the body. It is less clear, however, whether CR has a similar effect in the brain, particularly in the hippocampus, an area important for learning and memory processes that often are compromised in aging. In order to evaluate the effect of CR on synapses across lifespan, we quantified synapses stereologically in the middle molecular layer of the dentate gyrus (DG) of young, middle aged and old Fischer 344 x Brown Norway rats fed ad libitum (AL) or a CR diet from 4 months of age. The results indicate that synapses are maintained across lifespan in both AL and CR rats. In light of this stability, we addressed whether aging and CR influence neurotransmitter receptor levels by measuring subunits of NMDA (NR1, NR2A and NR2B) and AMPA (GluR1, GluR2) receptors in the DG of a second cohort of AL and CR rats across lifespan. The results reveal that the NR1 and GluR1 subunits decline with age in AL, but not CR rats. The absence of an aging-related decline in these subunits in CR rats, however, does not arise from increased levels in old CR rats. Instead, it is due to subunit decreases in young CR rats to levels that are sustained in CR rats throughout lifespan, but that are reached in AL rats only in old age.

Caloric restriction eliminates the aging-related decline in NMDA and AMPA receptor subunits in the rat hippocampus and induces homeostasis.

Caloric restriction (CR) extends life span and ameliorates the aging-related decline in hippocampal-dependent cognitive function. In the present study, we compared subunit levels of NMDA and AMPA types of the glutamate receptor and quantified total synapses and multiple spine bouton (MSB) synapses in hippocampal CA1 from young (10 months), middle-aged (18 months), and old (29 months) Fischer 344xBrown Norway rats that were ad libitum (AL) fed or caloric restricted (CR) from 4 months of age. Each of these parameters has been reported to be a potential contributor to hippocampal function. Western blot analysis revealed that NMDA and AMPA receptor subunits in AL animals decrease between young and middle age to levels that are present at old age. Interestingly, young CR animals have significantly lower levels of glutamate receptor subunits than young AL animals and those lower levels are maintained across life span. In contrast, stereological quantification indicated that total synapses and MSB synapses are stable across life span in both AL and CR rats. These results indicate significant aging-related losses of hippocampal glutamate receptor subunits in AL rats that are consistent with altered synaptic function. CR eliminates that aging-related decline by inducing stable NMDA and AMPA receptor subunit levels.

Synaptophysin and insulin-like growth factor-1 immunostaining in the central nucleus of the inferior colliculus in adult ferrets following unilateral cochlear removal: a densitometric analysis.

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.

Spatial learning and memory deficits after whole-brain irradiation are associated with changes in NMDA receptor subunits in the hippocampus.

Whole-brain irradiation is used for the treatment of brain tumors, but can it also induce neural changes, with progressive dementia occurring in 20-50% of long-term survivors. The present study investigated whether 45 Gy of whole-brain irradiation delivered to 12-month-old Fischer 344 x Brown Norway rats as nine fractions over 4.5 weeks leads to impaired Morris water maze (MWM) performance 12 months later. Compared to sham-irradiated rats, the irradiated rats demonstrated impaired MWM performance. The relative levels of the NR1 and NR2A but not the NR2B subunits of the NMDA receptor were significantly higher in hippocampal CA1 of irradiated rats compared to control rats. No significant differences were detected for these NMDA subunits in CA3 or dentate gyrus. Further analysis of CA1 revealed that the relative levels of the GluR1 and GluR2 subunits of the AMPA receptor and synaptophysin were not altered by whole-brain irradiation. In summary, a clinically relevant regimen of fractionated whole-brain irradiation led to significant impairments in spatial learning and reference memory and alterations in the relative levels of subunits of the NMDA, but not the AMPA, receptors in hippocampal CA1. These findings suggest for the first time that radiation-induced cognitive impairments may be associated with alterations in glutamate receptor composition.

Growth hormone and IGF-I modulate local cerebral glucose utilization and ATP levels in a model of adult-onset growth hormone deficiency.

Decreases in plasma IGF-I levels that occur with age have been hypothesized to contribute to the genesis of brain aging. However, support for this hypothesis would be strengthened by evidence that growth hormone (GH)/IGF-I deficiency in young animals produces a phenotype similar to that found in aged animals. As a result, we developed a unique model of adult-onset GH/IGF-I deficiency by using dwarf rats specifically deficient in GH and IGF-I. The deficiency in plasma IGF-I is similar to that observed with age (e.g., 50% decrease), and replacement of GH restores levels of IGF-I to that found in young animals with normal GH levels. The present study employs this model to investigate the effects of circulating GH and IGF-I on local cerebral glucose utilization (LCGU). Analysis of LCGU indicated that GH/IGF-I-deficient animals exhibit a 29% decrease in glucose metabolism in many brain regions, especially those involved in hippocampally dependent processes of learning and memory. Similarly, a high correlation between plasma IGF-I levels and glucose metabolism was found in these areas. The deficiency in LCGU was not associated with alterations in GLUT1, GLUT3, or hexokinase activity. A 15% decrease in ATP levels was also found in hippocampus of GH-deficient animals, providing compelling data that circulating GH and IGF-I have significant effects on the regulation of glucose utilization and energy metabolism in the brain. Furthermore, our results provide important data to support the conclusion that deficiencies in circulating GH/IGF-I contribute to the genesis of brain aging.

Synaptogenesis in the inferior colliculus of the pre-hearing postnatal ferret.

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.

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.

Caloric restriction does not reverse aging-related changes in hippocampal BDNF.

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.

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.

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.

Quantitative measurement of afferent layers in the ferret inferior colliculus: DNLL projections to sublayers.

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.