Ionizing radiation-induced alterations in the electrophysiological properties of Aplysia sensory neurons

Neuroscience Letters 268 (1999) 45±48

Ionizing radiation-induced alterations in the electrophysiological properties of Aplysia sensory neurons A.L. Clatworthy a,*, F. Noel b, E. Grose a, M. Cui a, P.J. To®lon b, c a Department of Biology, University of North Carolina at Charlotte, 9201 University City Boulevard Charlotte, NC 28223, USA Department of Experimental Radiation Oncology, UT. M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA c Department of Neurosurgery, UT. M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA

b

Received 28 January 1999; received in revised form 9 April 1999; accepted 15 April 1999

Abstract It is clear that ionizing radiation can alter neuronal function. Recently it has been suggested that radiation can directly in¯uence neurons and/or the neuronal microenvironment. We have developed a simple in vitro model system utilizing the marine mollusc Aplysia californica to test this hypothesis. We show that ionizing radiation at doses of 5, 10 or 15 Gy produces complex effects on the electrophysiological properties of a population of Aplysia nociceptive sensory neurons at 24 and 48 h post irradiation. These results add support to the notion that ionizing radiation can directly in¯uence neurons and/or the neuronal microenvironment. Furthermore, they demonstrate that Aplysia may be used as a useful model system to study radiation-induced neuronal plasticity. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Invertebrate sensory systems; Ionizing radiation; Aplysia; Electrophysiology; Hyperexcitability; Injury

Radiation-induced neurological injury is a debilitating complication for some cancer patients undergoing radiotherapy. Both acute and chronic neurotoxic effects have been reported following exposure of the brain to relatively low doses of ionizing radiation [1,2]. In animal models, electrophysiological changes have been reported in a variety of brain regions within hours of irradiation at doses of 10 Gy or less [14]. Mechanisms underlying radiation-induced neuronal alterations are poorly understood, and multiple factors are likely to contribute. Radiation in¯uences the systemic vasculature and the microvasculature in the brain resulting in decreased systemic blood pressure and a reduction in cerebral blood ¯ow [13]. Furthermore, radiation can compromise the integrity of the blood±brain barrier and progressive perturbations in cerebral energy metabolism have also been reported following whole brain irradiation in the therapeutic range [3,12]. Cultured astrocytes and microglial cells exposed to radiation produce TNF alpha which could potentially effect a variety of cell types [5]. Recent reports of alterations in neuronal function in irradiated hippocampal slices [15] raise the interesting possibility that some of the acute effects of radiation on neural * Corresponding author. Tel.: 11-704-547-4060; fax: 11-704547-3128. E-mail address: [email protected] (A.L. Clatworthy)

function may be mediated, at least in part, by a direct effect on neurons or the neuronal microenvironment. Additional support comes from studies demonstrating an inhibitory effect of ionizing radiation on voltage-sensitive calcium and sodium channels in rat brain synaptosomes [10]. The objective of the present experiments is use a simple systems approach to further explore the hypothesis that radiation induced alterations in neuronal function are mediated at least in part by a direct effect on neurons and/or the neuronal microenvironment. The marine mollusc Aplysia californica was chosen as the simple model system for these studies. Aplysia neurons are large and easily accessible for intracellular recording. Moreover, these neurons have been well characterized at functional, cellular and molecular levels. Furthermore, the nervous system of Aplysia can be removed and maintained in cell culture for several days allowing examination of more long-term effects of radiation on neuronal function in the absence of complicating systemic factors. Aplysia californica (150±250 g) supplied by Alacrity Marine (Redondo Beach, CA) were kept in arti®cial seawater (ASW) at 15±178C and fed seaweed (Sushinori). Experiments were performed using an isolated pleural-pedal ganglion preparation (Fig. 1). Dissections were performed after injecting the animal with isotonic MgCl2 solution equivalent to approximately 50% of the body weight.

0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 06 9- 5

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A.L. Clatworthy et al. / Neuroscience Letters 268 (1999) 45±48

Fig. 1. Schematic diagram of right pedal-pleural ganglia. Homologous clusters of sensory neurons are located on the ventrocaudal surface of the left pleural ganglion. Each sensory neuron sends an axon out to the periphery through an identi®ed ipsilateral pedal nerve. Left or right pedal-pleural ganglia were chosen randomly to receive the radiation treatment. The contralateral ganglia were not irradiated and served as internal controls. The experimenter was blind as to the treatment of each ganglion.

Right and left pleural-pedal ganglia and their associated pedal nerves were dissected out of the animal and the pedal commisure joining the two sides was cut (Fig. 1). The ganglia were pinned to the ¯oor of separate sylgard coated dishes ®lled with 3 ml ASW. One randomly selected side was exposed to a single dose of g radiation using a cesium 137 source at 5, 10 or 15 Gy and a dose rate of 3.9 Gy per min. The experimenter was blind as to the side that was irradiated. For preparations that were recorded 1 or 2 days after irradiation, the ganglia were placed in culture medium described in detail in Ref. [9]. Cultures were maintained in the dark at 158C. Immediately before recording, each pleural ganglion was desheathed in a solution containing equal volumes of isotonic MgCl2 solution and ASW. Intracellular recordings were made in ASW using glass capillary microelectrodes (10±20 MV ) ®lled with 3 M potassium acetate. Sensory neurons were examined sequentially, alternating between left and right sides and were paired by location in the ganglion. The protocol used to measure the electrophysiological properties of the sensory neurons is described in detail in [7]. Brie¯y, 20 ms intracellular depolarizing pulses were used to measure sensory spike threshold, amplitude and afterhyperpolarization. Two-millisecond pulses were used to measure spike duration. Excitability was tested by counting the number of spikes ®red in response to a 1 s depolarizing pulse at 1.25£ and 2.5£ the threshold determined with a 20 ms pulse. Input resistance was monitored by measuring

the change in potential elicited by injecting a standard 1 s, 0.5 nA intracellular hyperpolarizing pulse. Statistical comparisons between irradiated and non-irradiated sensory neurons were made with two-tailed paired t-tests averaging data from eight cells per side in each animal. In the ®rst series of experiments, the effects of 5, 10 and 15 Gy ionizing radiation on sensory spike threshold, amplitude, AHP, duration and input resistance were determined 24 h after exposure. In addition, the impact of radiation on the number of spikes elicited by a 1 s depolarizing pulse was assessed. Twenty-four hours after exposure to 5 Gy ionizing radiation, there were no signi®cant alterations in the electrophysiological properties of irradiated sensory neurons compared with contralateral, control sensory neurons. There was a trend for action potential duration recorded from irradiated sensory neurons to be longer than that recorded from contralateral control cells (4:45 ^ 0:32 vs. 3:72 ^ 0:24 ms; P ˆ 0:06; n ˆ 4 animals, 32 cell pairs). Twenty-four hours after exposure to 10 Gy ionizing radiation, sensory spike threshold recorded from irradiated sensory neurons was signi®cantly higher than spike threshold recorded from control cells (0:77 ^ 0:05 vs. 0:63 ^ 0:02 mA; P ˆ 0:02; n ˆ 5 animals, 39 cell pairs). There were no signi®cant differences between the electrophysiological properties of irradiated and control sensory neurons 24 h after exposure to 15 Gy ionizing radiation (n ˆ 4 animals, 31 cell pairs). In a second series of experiments, the studies described above were repeated but 48 h were allowed to elapse before assessing sensory neuron excitability. Forty-eight hours after exposure to 5 Gy ionizing radiation, the number of spikes elicited by a 1 s depolarizing pulse at 1.25 £ spike threshold was signi®cantly greater compared to contralateral, control cells (1:97 ^ 0:19 vs. 1:47 ^ 0:13 spikes;

Fig. 2. Examples of the response of two sensory neurons to a 1 s depolarizing pulse at (A) 1.25£ and (B) 2.5£ spike threshold. Recordings were made from a control, non-irradiated neuron and a neuron from the contralateral ganglion that was paired by location in the ganglion. Experimental ganglia had been exposed 48 h previously to 10 Gy ionizing radiation. Note the larger number of spikes elicited by the standard stimulus in the sensory neuron that had been irradiated.

A.L. Clatworthy et al. / Neuroscience Letters 268 (1999) 45±48

47

Table 1 Effects of 5, 10 and 15 Gy ionizing radiation on the excitability of Aplysia sensory neurons at (A) 24 and (B) 48 h post irradiation a Threshold

Amplitude

(A) 24-h post irradiation 5 Gy 1 2 10 Gy 1** 2 15 Gy 2 2 (B) 48-h post irradiation 5 Gy nc nc 10 Gy 2** 2 15 Gy 2* 1

AHP

1 s pulse @ 1.25£ threshold

1 s pulse @ 2.5£ threshold

Duration

Input resistance

Number of animals

Number of cell pairs

1 2 2

2 1 1

2 1 2

1* 2 2

1 1 2

4 5 4

32 39 31

1 1 1

1** 1** 2

1 1** 1

1 1 1

2 1 1**

4 4 4

32 32 32

a 1 or 2 represent experimental values with respect to control values. For example, 24 h following exposure to 10 Gy ionizing radiation, irradiated sensory neurons had a signi®cantly higher threshold compared with control neurons. In contrast, 48 h post irradiation, irradiated sensory neurons had a signi®cantly lower threshold compared with control non-irradiated neurons. nc, not changed; **Statistically signi®cant (two-tailed paired t-test; P , 0:05); *trend (two-tailed paired t-test; P , 0:1).

P ˆ 0:03; n ˆ 4 animals, 32 cell pairs). Forty-eight hours after exposure to 10 Gy ionizing radiation, sensory neurons showed a signi®cant decrease in sensory spike threshold (0:56 ^ 0:06 vs. 0:73 ^ 0:08 mA; P ˆ 0:05; n ˆ 4 animals, 32 cell pairs) and a signi®cant increase in the number of spikes elicited by a 1 s depolarizing pulse at both 1.25£ and 2.5£ spike threshold (2:06 ^ 0:24 vs. 1:47 ^ 0:16 spikes; and 5:38 ^ 0:38 vs. 3:91 ^ 0:36 spikes; P ˆ 0:02 and P ˆ 0:003 respectively; n ˆ 4 animals, 32 cell pairs). An example of the effect of 10 Gy radiation on the number of spikes elicited by a standard depolarizing pulse at 48 h is illustrated in Fig. 2. Sensory neurons exposed to 15 Gy ionizing radiation showed a signi®cant increase in input resistance 48 h after exposure (42:66 ^ 4:61 vs. 29:56 ^ 3:91 MV ; P ˆ 0:04; n ˆ 4 animals; 32 cell pairs). There was a trend for spike threshold to be lower in irradiated cells compared to control cells (0:7 ^ 0:06 vs. 0:91 ^ 0:09 mA; P ˆ 0:07). The effects of 5, 10 and 15 Gy ionizing radiation on sensory excitability at 24 and 48 h post irradiation are summarized in Table 1. Signi®cant radiation-induced effects on sensory excitability were obtained within some individual animals and this, coupled with the fact that the number of ganglion pairs compared in each study was small (usually four pairs, see Table 1) suggests that the reported effects of radiation on sensory excitability are indeed real. The present results demonstrate that ionizing radiation can have complex effects on the electrophysiological properties of Aplysia sensory neurons. Twenty-four hours after irradiation there were few signi®cant changes in sensory excitability. There was a tendency for 5 Gy radiation to produce a lengthening of the sensory spike, 10 Gy produced a signi®cant increase in spike threshold while 15 Gy produced no signi®cant effects. In contrast, 48 h after irradiation, the signi®cant alterations in sensory function re¯ected an increase in excitability. Thus, following 5 Gy irradiation, there was a signi®cant increase in the number of spikes elicited by a 1 s depolarizing pulse. In contrast to the effects of 10 Gy ionizing radiation 24 h after irradiation, 48 h after exposure to 10 Gy, a signi®cant decrease in spike

threshold was recorded. In addition, a signi®cant increase in the number of spikes elicited by the 1 s depolarizing pulse was recorded. There was a trend for spike threshold to be lower following 15 Gy ionizing radiation. The present studies are not the ®rst to utilize Aplysia as a model system to determine the effects of radiation on neuronal function [4,16,20] although they are the ®rst to look at the long-term effects (24±48 h) of relatively low doses of radiation on a population of identi®ed sensory neurons. Unfortunately, the wide range of doses employed and the different neuronal populations sampled in each of the previous studies make direct comparison with the present studies impossible. At present we do not know the mechanisms underlying the radiation-induced alterations in sensory neuron function. It is interesting that the increase in sensory neuron excitability recorded 48 h after irradiation is qualitatively similar to injury-induced sensory hyperexcitability recorded in Aplysia following axonal crush [7,19]. It is possible that commonalties could exist between mechanisms underlying injury-induced and radiation-induced alterations in sensory function. A persistent decrease in K conductances or an increase in the density of voltage sensitive Na channels could explain the increases in sensory neuron excitability. The Aplysia preparation lends itself well to the kinds of studies required to determine the ionic mechanisms underlying radiation-induced alterations in sensory excitability. We cannot rule out the possibility that the effects may be mediated at least in part at the level of the neuronal microenvironment which would include microglial-like cells that have been identi®ed in the connective tissue sheath surrounding the cell bodies and axons in a related mollusc [18]. Although the connective tissue sheath surrounding the pleural ganglion was removed in the present studies, the sheath surrounding the pedal ganglion and the pedal nerves was intact. Recent reports demonstrating that cultured astrocytes and microglial cells can be stimulated by radiation to produce TNFa [5] are interesting because there is evidence for cytokine-induced modulation of neuronal function in

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A.L. Clatworthy et al. / Neuroscience Letters 268 (1999) 45±48

both molluscan and mammalian systems [8,11,17]. The question of a direct versus indirect effect could be addressed by testing the effects of ionizing radiation on homogenous populations of cultured dissociated VC sensory cells. As these preparations are devoid of connective tissue sheath, any effect of ionizing radiation would have to be mediated by a direct effect on the neurons. The population of sensory neurons described in this study has been characterized as wide dynamic range nociceptors [6]. A signi®cant increase in the excitability of these sensory would be functionally similar to hyperalgesia and allodynia in mammalian systems. The types of studies outlined in the present paper may therefore also provide clues for understanding mechanisms underlying radiation-induced neuropathies that have been reported in some cancer patients following radiotherapy, the initial symptoms of which are usually sensory in nature and include pain. In conclusion, a detailed understanding of fundamental mechanisms underlying radiation-induced alterations in neuronal function is critical for the future study and development of neuroprotective agents that could be used in conjunction with conventional radiotherapy. The present studies demonstrate that the marine mollusc Aplysia may indeed serve as a useful model system to address questions related to radiation-induced neuronal plasticity and suggest that further studies are warranted. For example, the well characterized synaptic connections in Aplysia could be utilized to study radiation-induced alterations in the modulation of synaptic function. Moreover, the relative simplicity of the Aplysia model system lends itself well to experiments designed to understand cellular mechanisms underlying radiation-induced neuronal plasticity. These studies were supported by Grant R29 MH53559 from the National Institute of Mental Health to A.L.C. We thank two anonymous reviewers for helpful comments on an earlier version of this paper. [1] Al-Mefty, O., Kersh, J.E., Routh, A. and Smith, R.R., The long term side effects of radiation therapy for benign brain tumors in adults. J. Neurosurg., 73 (1990) 502±512. [2] Anno, G.H., Baum, S.J., Withers, H.R. and Young, R.W., Symptomology of acute radiation effects in humans after exposure to doses of 0.5±30 Gy. Health Phys., 56 (1989) 821± 838. [3] d'Avella, D., Cicciarello, R., Gagliardi, M.E., Albiero, F., Mesiti, M., Russi, E., D'Aquino, A. and Tomasello, F., Progressive perturbations in cerebral energy metabolism after experimental whole-brain radiation in the therapeutic range. J. Neurosurg., 81 (1994) 774±779. [4] Carpenter, D.O., Gaubatz, G., Willis, J.A. and Severance, R.,

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