Conceived and designed the experiments: AC JRF SR. Performed the experiments: AC AA BA. Analyzed the data: AC JRF. Contributed reagents/materials/analysis tools: AC JRF SR BA. Wrote the paper: AC AA SR JRF.
The authors have declared that no competing interests exist.
Therapeutic irradiation of the brain is a common treatment modality for brain tumors, but can lead to impairment of cognitive function. Dendritic spines are sites of excitatory synaptic transmission and changes in spine structure and number are thought to represent a morphological correlate of altered brain functions associated with hippocampal dependent learning and memory. To gain some insight into the temporal and sub region specific cellular changes in the hippocampus following brain irradiation, we investigated the effects of 10 Gy cranial irradiation on dendritic spines in young adult mice. One week or 1 month post irradiation, changes in spine density and morphology in dentate gyrus (DG) granule and CA1 pyramidal neurons were quantified using Golgi staining. Our results showed that in the DG, there were significant reductions in spine density at both 1 week (11.9%) and 1 month (26.9%) after irradiation. In contrast, in the basal dendrites of CA1 pyramidal neurons, irradiation resulted in a significant reduction (18.7%) in spine density only at 1 week post irradiation. Analysis of spine morphology showed that irradiation led to significant decreases in the proportion of mushroom spines at both time points in the DG as well as CA1 basal dendrites. The proportions of stubby spines were significantly increased in both the areas at 1 month post irradiation. Irradiation did not alter spine density in the CA1 apical dendrites, but there were significant changes in the proportion of thin and mushroom spines at both time points post irradiation. Although the mechanisms involved are not clear, these findings are the first to show that brain irradiation of young adult animals leads to alterations in dendritic spine density and morphology in the hippocampus in a time dependent and region specific manner.
Cranial irradiation is an essential therapeutic tool in the treatment of primary and secondary malignancies, but can be associated with a risk for adverse side effects, including cognitive dysfunction
The hippocampus plays a crucial role in learning and memory
The formation of long-term memory relies on modulation of synaptic connections in response to neuronal input. This plasticity requires coordinated activity-dependent synthesis of specific mRNAs and proteins that facilitate molecular and structural changes at the synapse
The purpose of the present study was to determine the temporal effects of cranial irradiation on spine density and morphology in the dendrites of granule neurons of dentate gyrus as well as pyramidal neurons of CA1 area of the hippocampus. Since pyramidal neurons typically consist of apical and basal dendrites which differ in their connectivities, biophysical characteristics and long term potentiation induction and expression mechanisms
The study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the University of California, San Francisco (UCSF) Institutional Animal Care and Use Committee (IACUC).
A total of 20 two month old male C57BL/6J mice (n = 5/group) were used for this study. Animals were purchased from a commercial vendor (Jackson Laboratory, Bar Harbor, ME) and were group housed (5 mice/cage) throughout the study. Mice were maintained on a 12-h light-dark cycle and were provided food and water ad libitum. All efforts were made to minimize suffering of the animals.
For irradiation, mice were anesthetized using an i.p injection of ketamine (70 mg/kg) and medetomidine (0.5 mg/kg). Mice were placed prone, 16.3 cm from a cesium-137 source (J.L. Shepherd & Associates, San Fernando, CA) and shielded with an iron collimator that limited the irradiation to a 1 cm wide vertical beam
For spine analyses, Golgi staining was performed using the FD Rapid Golgi Stain Kit (FD Neurotechnologies, Baltimore, MD)
Spine analyses were conducted blind to the experimental conditions on coded Golgi impregnated brain sections containing the dorsal hippocampus. Spines were examined on dendrites of DG granule neurons (
(A) A dentate gyrus granule neuron and (B) a CA1 pyramidal neuron illustrating basal dendrites in the stratum oriens and apical dendrites in the stratum radiatum. Scale bar = 50 µm.
The neurons that satisfied the following criteria were chosen for analysis in each of the experimental groups: i) presence of untruncated dendrites; ii) consistent and dark Golgi staining along the entire extent of the dendrites; and iii) relative isolation from neighboring neurons to avoid interference with analysis
On the basis of morphology, spines were classified into the following categories: i) Thin: spines with a long neck and a visible small head; ii) Mushroom: big spines with a well-defined neck and a very voluminous head; and iii) Stubby: very short spines without a distinguishable neck and stubby appearance
(A) Thin (B) Mushroom and (C) Stubby spines (arrows). Scale bar = 5 µm.
To acquire images for spine analysis, the dendritic segments were imaged under brightfield illumination on a Zeiss Axioimager microscope with a 63x oil immersion objective. Spine analyses were based on the method of Margarinos et al
Data were expressed as Mean ± SEM. An unpaired two-tailed
In the DG, there were significant reductions in spine density at both 1 week (11.9%, p<0.05) and 1 month (26.9%, p<0.001) after irradiation (
Brain irradiation induced time and region specific changes in the numbers of dendritic spines/10 µm in the DG granule neurons as well as pyramidal neurons in the CA1 region. The open bars represent unirradiated animals and the dark bars represent mice irradiated with 10 Gy of gamma rays. Each bar represents the mean value of 5 animals and error bars are SEM. *represents a p<0.05, and **represents p<0.001.
In the DG, 1 week after irradiation, there was a significant increase (9.6%, p<0.05) in the proportion of thin spines while at 1 month the difference was not statistically significant (
Brain irradiation induced time and region specific changes in the proportion of spine morphological sub types in the DG granule neurons as well as pyramidal neurons from the CA1 region. The open bars represent unirradiated animals and the dark bars represent mice irradiated with 10 Gy of gamma rays. Each bar represents the mean value of 5 animals and error bars are SEM. *represents a p<0.05, and **represents p<0.001.
In the CA1 basal dendrites, irradiation significantly increased the proportion of thin spines at 1 week (11.7%, p<0.05) but no changes were observed at the later time point (
Despite the fact that no changes in spine density were observed in the CA1 apical dendrites at either time point, there were significant differences in both thin and mushroom spine morphology between the sham and irradiated groups (
The present study demonstrated that brain irradiation altered spine density as well as the proportion of morphological subtypes in the dendrites of DG granule neurons and basal dendrites of CA1 pyramidal neurons in a time dependent manner. While there was a gradual decrease in spine density in the DG over time, spine density in the CA1 basal dendrites decreased at 1 week post irradiation with a trend toward recovery at 1 month. Additionally, in the CA1 apical dendrites, irradiation altered spine morphology without any change in spine density at both 1week and 1month post irradiation. To our knowledge, these results are the first to demonstrate that, in young adult mice, cranial irradiation affects dendritic spine density and morphology in the hippocampus in a temporal and region specific manner.
The maintenance of normal brain function is dependent on the establishment and efficient maturation of synaptic circuits
Dendritic spines are the primary recipients of excitatory input in the CNS, and changes in spine density and morphology can account for functional differences at the synaptic level
Golgi staining is a reliable and sensitive method for revealing the morphological details of individual neurons, especially dendritic spines
A number of factors might account for the observed differences in spine density between the two hippocampal subregions. Numerous studies have demonstrated that spine density is regulated by glutamatergic transmission and glutamate receptor subtypes located on dendritic spine heads
In our earlier studies using the same dose of radiation in the same strain of mice, we found increased numbers of activated microglia in the DG 1 week, which became significant at 2 months post irradiation
In contrast to DG and CA1 basal dendrites, irradiation did not alter spine density in CA1 apical dendrites. Differential vulnerability between basal and apical dendrites due to exogenous or endogenous factors has been reported in the literature although the mechanisms involved are not clear. For instance, Santos et al
One of the most remarkable features of dendritic spines is their morphological diversity
Our data showed that in both DG and CA1 basal dendrites, spines characterized by the mushroom morphology were particularly affected by radiation exposure. Mushroom spines have larger postsynaptic densities
Whereas radiation exposure led to decrease in the fraction of mushroom spines, a marked increase in the proportion of stubby spines were observed in both DG and CA1 basal dendrites 1 month post irradiation. Although less is known about these stubby structures, they have been shown to predominate early in postnatal development
Despite the fact that no change in spine density was observed in the apical dendrites of CA1 neurons after irradiation, significant differences in thin and mushroom spine morphology were observed between the sham and irradiated groups. It is noteworthy that contrary to what was observed in DG and CA1 basal dendrites, irradiation led to significant decreases in the percentages of thin spines after irradiation and a significant increase in mushroom spines. The length of the spine neck seems to be a key regulator of spinodendritic Ca2+ signaling and of the transmission of membrane potentials
The functional implications of the observed radiation effects on dendritic spines at the two hippocampal sub regions are not yet clear. Additionally, if or how these radiation-induced alterations may relate to the behavioral
In conclusion, to the best of our knowledge the present report provides the first evidence that in young adult mice, cranial irradiation causes alteration in spine density and morphology in the hippocampus in a time dependent and region specific manner. Since loss of dendritic spines or structural reorganizations of spines play an important role in learning and memory, the observed changes suggest a disruption of neural circuitry that might play a role in radiation induced cognitive impairment.
The authors would like to thank Kirsten Eilertson, Ph.D at the Gladstone Bioinformatics Core for guidance with statistical analysis.