In the present study, we systemically examined the effects of space-relevant, low-dose proton irradiation on local synaptic circuits within the hippocampus, a brain area centrally important for some of the cognitive tasks affected by radiation exposure (Britten et al.
2012,
2014; Bellone et al.
2015). The key findings are: (1) Proton irradiation resulted in a large, persistent potentiation of action potential-dependent GABA release from CB
1BCs onto CA1 PCs, without alterations in the axonal or dendritic morphology or intrinsic excitability of these interneurons; (2) The mechanism of the radiation-induced increase in GABA release from CB
1BCs was a significant decrease in the CB
1-mediated control of GABA release, associated with depressed 2-AG levels; (3) The effects of irradiation on GABA release were cell type-specific, since proton exposure did not alter GABA release from PVINs; (4) Local excitatory circuits were also modulated by radiation, as indicated by a proton-induced enhancement of the connection probability between PCs and PVINs, without significant increases in the PC to CB
1BC connections. Therefore, we have demonstrated that energetic solar particles selectively alter local GABAergic inhibition and glutamatergic excitation in the hippocampus.
Cellular-synaptic bases of persistent cognitive impairments associated with radiation exposures relevant to interplanetary travel
With recent advances in space exploration, the possibility of human interplanetary travel no longer seems like science fiction. Before sending humans on such long-term voyages in space, the immediate and long-term health risks caused by exposure to space radiation fields must be carefully evaluated. Decades of clinical experience in the management of brain tumors have revealed the adverse effects of cranial irradiation on cognition (Meyers
2000; Butler et al.
2006). While clinical irradiation scenarios are clearly distinct (i.e., in terms of radiation dose and type) from those in space, significant recent work using rodent models has now corroborated that very low doses of charged particles can disrupt cognition using a variety of behavioral tasks (Britten et al.
2012; Lonart et al.
2012; Parihar et al.
2015a).
In addition, these decreases in cognitive performance were associated with structural changes in dendrites and alterations in key synaptic proteins (Parihar and Limoli
2013; Parihar et al.
2014,
2015a; Allen et al.
2015; Chmielewski et al.
2016). However, our understanding of how charged particles may impact specific excitatory and inhibitory circuits in the brain has been limited. In this paper, we studied the effects of the space relevant dose of 0.5 Gy on key hippocampal microcircuits. We chose to focus on the effect of protons, given that they constitute the vast majority of charged particles in space (Cucinotta et al.
2014; Nelson
2016). We carried out our experiments in the hippocampus, because it plays crucial roles in cognitive tasks that include memory consolidation and spatial navigation. Hippocampal perisomatic inhibition is mediated by CB
1BCs and the numerically dominant PV basket cells within the PVIN class. These cells are specialized to form multiple synaptic contacts on the somata and proximal dendrites of hundreds of postsynaptic PCs. Because of the proximity of their inhibitory output synapses to the AP initiation site located on the initial segment of PCs, perisomatically projecting interneurons are in a strategic position to control hippocampal network output. Importantly, there is a strict division of labor between CB
1BCs and PVINs. Hippocampal PVINs receive large amounts of excitatory inputs, fire fast and non-accommodating APs, have fast membrane time constants, and release GABA in synchrony with presynaptic APs (Armstrong and Soltesz
2012). Thus, PVINs can function as the precisely timed inhibitory elements of the hippocampal circuit and play key roles as time-keepers in the generation of distinct behaviorally relevant network rhythms, including theta and gamma oscillations and sharp wave ripples. In contrast, CB
1BCs are the more modifiable elements of perisomatic inhibitory control (Armstrong and Soltesz
2012), since these cells express an especially large variety of receptors for various neuromodulators, including endocannabinoids. Pathological alterations in PVIN and CB
1BC properties and functions have been reported in a variety of neurological disorders, including epilepsy, schizophrenia, autism, and Huntington disease (Chen et al.
2003; Curley and Lewis
2012; Dvorzhak et al.
2013; Földy et al.
2013).
Here we have shown that the effect of proton irradiation has long-lasting, highly specific effects on hippocampal perisomatic inhibitory microcircuits, and that the radiation-induced plasticity involves not only the interneuronal inputs from CB
1PCs to PCs but also the excitatory innervation of PVINs by local CA1 PCs. The specificity of these effects indicate that proton irradiation does not indiscriminately affect the synaptic properties of neuronal circuits, in spite of the fact that the effect size was large, specifically, a more than sixfold increase in GABA release from CB
1BCs without any alteration in the release properties from PVINs. The specificity and magnitude of these persistent alterations in perisomatic inhibitory circuits are consistent with the marked alterations in cognitive performance after space-relevant doses of radiation (Lonart et al.
2012; Bellone et al.
2015). Indeed, given the reported roles of CB
1BCs in a variety of circuit functions, including the integration of synaptic inputs from a variety of local and long-distance sources, as well as in the modulation of the input–output gains of CA1 PCs during network activity and the expression of input-timing depending plasticity, the robust increase in GABA release from CB
1BCs after proton irradiation likely affects the assessment of the saliency of inputs arriving at the hippocampus from the entorhinal cortex (Armstrong and Soltesz
2012; Basu et al.
2013). In turn, the resulting lack of proper filtering of incoming salient information about the environment is expected to lead to aberrant memory consolidation. Similarly, PVINs are thought to be involved in the generation of oscillations in the hippocampal network, particularly the high frequency gamma oscillations, through their fast-spiking properties and their strong connectivity with PCs (Fuchs et al.
2007; Sohal et al.
2009; Holderith et al.
2011; Buzsáki and Wang
2012; Hu et al.
2014). Therefore, alterations to the excitatory inputs of PV cells after proton irradiation likely compromise the precise spike timing necessary for the generation of gamma oscillations, with downstream effects on associated cognitive functions, such as spatial memory, attention, and cognitive flexibility (Isaacson and Scanziani
2011; Buzsáki et al.
2012; Cho et al.
2015; Kim et al.
2016). These results indicate that irradiation-related pathological alterations of either CB
1BCs or PVINs could result in deficits in hippocampus-dependent cognitive function.
Our findings indicating selective radiation-induced alterations to inhibitory and excitatory synapses are in overall agreement with previous reports of both hypo- and hyperexcitable modifications in hippocampal circuits after space-relevant doses of radiation. For example, the present data showing increased GABA release from CB
1BCs and enhanced excitatory innervation of PVINs suggest an augmentation of perisomatic inhibition of PCs, in agreement with findings indicating hyperpolarized resting membrane potential and decreased input resistance of PCs after proton irradiation (Sokolova et al.
2015). However, perisomatic inhibition can modulate hippocampal excitability in complex ways (Armstrong and Soltesz
2012). For example, increased inhibitory inputs can cause paradoxical rebound spiking and increases in synchronized discharges in hippocampal circuits (Cobb et al.
1995; Chen et al.
2001). Space-relevant irradiation using low doses of protons or high-energy charged particles (e.g.,
28Si and
56Fe) generally resulted in an overall increase in hippocampal excitability as assessed by field EPSPs in the CA1 and the dentate gyrus (Vlkolinský et al.
2007; Marty et al.
2014; Rudobeck et al.
2014; Bellone et al.
2015). Similarly, reports indicating increases in postsynaptic density protein (PSD-95) expression, persistent sodium currents, and excitatory synaptic transmission are also consistent with hippocampal hyperexcitability caused by space-relevant irradiation (Parihar et al.
2014; Sokolova et al.
2015). Computational modeling—taking into account some of these proton-irradiation-induced complex alterations in cellular and synaptic excitability—suggested that a perturbation in behaviorally relevant theta-frequency oscillations may take place in hippocampal networks (Sokolova et al.
2015) that could partially underlie radiation-induced disturbances in cognitive performance.
Perturbation of the endocannabinoid system and neurological dysfunction
Our experiments identified that loss of CB
1-mediated tonic inhibitory control was a major factor underlying the marked upregulation of GABA release from CB
1BCs after proton irradiation. In the hippocampus, CB
1s are highly expressed on axon terminals of specific subtypes of GABAergic interneurons (e.g., CB
1BCs), as well as on subsets of excitatory terminals (Katona et al.
1999; Mackie
2005; Soltesz et al.
2015). The activity of presynaptic CB
1s exerts robust inhibition of GABA release (Neu et al.
2007; Hashimotodani et al.
2007; Lee et al.
2010; Kim and Alger
2010; Lee et al.
2015). Evidence is mounting that perturbations of tonic cannabinoid signaling occur in a variety of neurologic disorders including epilepsy, Fragile X syndrome, autism, schizophrenia and chronic ethanol exposure (Chen et al.
2003; Maccarrone et al.
2010; Curley and Lewis
2012; Dvorzhak et al.
2013; Földy et al.
2013; Tang and Alger
2015; Varodayan et al.
2016). Additionally, the knockout of β-neurexins, important cell adhesion molecules, leads to decreased tonic endocannabinoid signaling and results in impairment of contextual memory (Anderson et al.
2015), and genetic deletion of CB
1s from GABAergic interneurons results in hippocampus-dependent spatial memory impairments (Albayram et al.
2016). Therefore, our results indicating that proton irradiation leads to a large, long-term decrease in CB
1-mediated tonic inhibition of GABA release from CB
1BCs to PCs, accompanied by decreased levels of 2-AG, are consistent with the sensitivity of the endocannabinoid signaling system to a variety of perturbations, with significant consequences for circuit performance. Basal levels of 2-AG are tightly controlled by the activities of both diacylglycerol lipase-α (2-AG synthetic enzyme), which is highly localized on dendritic spines of PCs, and monoacylglycerol lipase (2-AG degrading enzyme), which is localized on excitatory and inhibitory presynaptic terminals as well as astrocytes (Soltesz et al.
2015; Lee et al.
2015). Therefore, the decreased levels of 2-AG following proton irradiation may be due to downregulated diacylglycerol lipase-α and/or upregulated monoacylglycerol lipase, and future investigations will be conducted to discriminate between these and potentially other possibilities.
In summary, our results demonstrate that space relevant doses of proton irradiation causes persistent, large and highly specific alterations to perisomatic inhibitory circuits. Given the crucial roles that the perisomatically projecting interneurons play in hippocampus-dependent cognitive tasks, these changes are likely to be mechanistically linked to the cognitive effects found after radiation exposure in a variety of settings including space travel. The specific nature of the radiation-induced changes in perisomatic circuits may also present future opportunities for designing novel therapeutic avenues targeting the versatile cannabinoid signaling system (Soltesz et al.
2015), as well as other molecular pathways that may be involved.