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 
. The hippocampus plays a key role in learning and memory processes 
and is particularly susceptible to the effects of ionizing irradiation 
. While irradiation has been shown to change the numbers of newly born neurons in the DG 
, data also exist showing changes associated with learning and memory that do not involve overt mature neuronal loss 
. This latter finding suggests that changes in structure and function of viable neuronal cells may play an important role in the development of cognitive deficits after irradiation, and highlights the potential importance of assessing critical structures such as dendritic spines.
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 
. Spine morphology can predict both spine stability and synaptic strength 
and findings from in vivo
models support the notion that structural plasticity of spines is related to learning and memory function 
. Spines also compartmentalize Ca2+
and other signaling components conferring specificity to changes in synaptic efficacy and protecting neurons from excitotoxicity 
. In light of the multiple spine functions, pathological changes in spine number and structure may have significant consequences for brain function, as has been shown in studies of stress, malnutrition, toxins and drugs of abuse 
Golgi staining is a reliable and sensitive method for revealing the morphological details of individual neurons, especially dendritic spines 
. One drawback to this technique is that it cannot be effectively combined with other staining techniques 
. Because the goal of the present study was only to address spine density and morphology and not to identify other cell types, we selected this method over other available techniques for spine analysis. The analysis of Golgi stained neurons showed that radiation exposure led to a gradual decrease in spine density in the DG over time. In contrast, spine density in the CA1 basal dendrites decreased at 1 week post irradiation with a trend toward recovery at 1 month. The observed reductions in spine density might indicate early signs of neuronal injury in the hippocampus following irradiation and also suggest that there is a time dependent vulnerability of the two hippocampal sub regions following radiation exposure.
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 addition, a series of in vitro
studies have shown that N-methyl-D-aspartic acid (NMDA) receptors mediate the destabilization of filamentous actin (f-actin) associated with dendritic spine loss 
. Although the effects of radiation on NMDA receptor dynamics on hippocampal sub-regions are not well understood, studies by Shi et al 
have shown differential changes in subunits of NMDA receptors in the hippocampal subfields following whole brain irradiation. Thus, it is tempting to speculate that the observed temporal differences in reduction in spine density between DG and CA1 basal dendrites may involve differential alterations of NMDA receptor mediated responses in these two areas following irradiation. Brain derived neurotrophic factor (BDNF) is another well characterized determinant of dendritic spine number and morphology 
. Regulation of BDNF and its receptor expression has been reported to be very sensitive to radiation in the hippocampus and such changes vary depending on time after irradiation 
. Therefore, it is also possible that radiation might differentially alter BDNF and its downstream signaling targets in the dendrites of dentate granule cells and CA1 basal dendrites which may account for the differential changes in spine density at these two regions as a function of time after irradiation.
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 
. Therefore, the gradual decrease in spine density over time observed in the DG () could be associated with an increase in microglial activation. Other investigators have recently shown that changes in dendritic spines are associated with alterations in microglia 
, an effect that may be associated with the release of soluble factors 
. Further studies are in progress to address the molecular mechanisms involved in the observed temporal differences in radiation induced alterations in spine density.
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 
reported that neonatal rats exposed repetitively to low doses of paroxon (a organophosphate-type cholinesterase inhibitor) lost dendritic spine selectively in basal dendrites with no changes in apical dendrites of CA1 pyramidal neurons. Moreover normal aging also results in decreases of the spine density on basal but not apical dendrites in C57BL/6 mice 
. Future studies will be required to evaluate the mechanistic basis of differential vulnerability in radiation induced reduction of spine density between CA1 basal and apical dendrites.
One of the most remarkable features of dendritic spines is their morphological diversity 
. The three categories studied here appear to have different functional properties, including activity induced changes in intracellular calcium concentration, glutamate receptor levels and perhaps new versus well established memory processing 
. Additionally, dendritic spine morphology has also been reported to affect the diffusion and compartmentalization of membrane associated proteins and expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors 
. Given this information, we assessed whether the proportions of each type of spine were altered in the DG and CA1 area following radiation exposure.
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 
which anchor more AMPA glutamate receptors and make these synapses functionally stronger 
. Mushroom spines are more likely to contain smooth endoplasmic reticulum, which can regulate calcium locally 
and spines that have larger synapses are also more likely to contain polyribosomes for local protein synthesis 
. Thus, the loss of mushroom spines as seen here may have a more profound effect on neuronal function than the loss of the other types of spines. Gao et al
has also recently reported that moderate traumatic brain injury in mice led to significant decrease in mushroom shaped spines indicating a reduction in number of synapses which was confirmed by synaptophysin staining.
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 
and to increase in mature hippocampal slices after synaptic transmission was blocked 
. It has also been reported that dopamine receptors are located on the spine neck in the perisynaptic space 
and stubby spines that lack a neck likely have abnormal distributions of dopamine receptors in this space 
. It can be speculated that a marked increase in the proportion of stubby spines by radiation exposure might therefore lead to some alterations in dopaminergic signaling. Because radiation has been reported to affect dopaminergic processes in the brain 
, such changes may have long-term consequences for radiation induced cognitive changes.
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 
. Thin spines maintain the structural flexibility to enlarge and stabilize after long term potentiation and can accommodate new, enhanced or recently weakened inputs, making them candidate ‘learning spines’ 
. By decreasing the proportion of learning spines, radiation may therefore decrease a neuron’s ability to form new synapses and changes in activity in the CA1 apical dendrites. Age related reductions in thin spines have been observed in rhesus monkeys, with cognitive performance inversely proportional to thin spine volume 
. Although the reason for the corresponding increase in mushroom spines in these dendrites is not clear, it might represent a homeostatic mechanism to compensate for the reduction of the learning spines.
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 
, cellular 
observed at the same dose and/or time used here, remains to be determined.
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.