Running Head: Hippocampal Neurogenesis and Pain Behavior Alterations in Hippocampal Neurogenesis and Pain Behavior in Mice: An Experimental Study

As living condition has been shown to affect rates of neurogenesis, the current study was designed to examine the relationship between neurogenesis and pain behavior, particularly stressinduced analgesia (SIA) and tonic pain. Mice received daily injection stress and were placed in group or isolated housing conditions, with or without access to a running wheel in order to differentially manipulate neurogenesis. Both males and females were used in this study to investigate sex differences involved. Animals were tested with hotplate and tail withdrawal tests before and after restraint stress to examine SIA (experiment 1), and with a subcutaneous formalin injection to assess changes in tonic pain—particularly in the late phase (experiment 2). Group housed runners were expected to have increased neurogenesis, decreased SIA and increased late phase formalin pain; however, findings did not support this hypothesis. In experiment 1, significant differences in overall acute pain were observed, depending on housing and running. In experiment 2, overall formalin pain behavior was influenced by housing, while running differentially influenced pain behaviors in each phase. Significant changes in neurogenesis were not observed, which--along with a number of confounding variables--may have influenced inconsistent findings. Hippocampal Neurogenesis and Pain Behavior 4 Introduction After much controversy over its initial discovery, neurogenesis in the adult mammalian brain, particularly in the hippocampus and olfactory bulb, has become a generally accepted phenomenon. Neurogenesis has been widely studied to explore its effects on memory and learning. Investigators have manipulated variables such as living environment, physical activity levels, and hormonal levels to observe how these factors influence neurogenesis, and, in turn, learning and memory. However, the relationship between neurogenesis and pain has not received as much attention. Various studies have suggested that learning and various types of pain behavior may result from common mechanisms involving neuronal plasticity. Therefore, neurogenesis may have effects on pain that are parallel to those involving learning and memory. The current study attempted to explore this relationship. In 2006, Stranahan, Khalil, and Gould found that different social environments and levels of physical activity have significantly different effects on neurogenesis; certain conditions increase neurogenesis, while others decrease its occurrence. We will use manipulations similar to theirs to place mice in differentially neurogenesis-promoting conditions and investigate the relationship between neurogenesis and pain behavior. We will use both male and female mice to examine any sex differences that may exist. In particular, we will be investigating late-phase formalin pain behavior—particularly the late phase plastic changes—and stress-induced analgesia. Pain The word “pain” has been used to define stimuli, responses, sensations, and perceptions experienced by an individual. The International Association for the Study of Pain (2007) operationalizes pain as an “unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage”( http://www.iasp-pain.org, Pain section ). The sensation of pain serves to indicate current or potential tissue damage, as well as evoke responses that protect the body from harm. (Sternbach 1969). Hippocampal Neurogenesis and Pain Behavior 5 The sensation of pain is adaptive; as it is naturally aversive, pain acts as a warning to avoid harm. Thus, animals have evolved nervous systems to understand the information they receive from the environment and to determine whether stimuli are innocuous or noxious (tissue damaging). They can then use this information to decide to withdraw from and avoid potentially dangerous stimuli. Additionally, the pain system has evolved methods for altering an individual’s pain sensation; for example, increasing one’s sensitivity to pain for a more rapid response and avoidance of noxious stimuli. In humans, the experience of pain involves the sensation of a noxious stimulus, followed by physiological and affective or emotional responses (Sternberg 2007). In this way, there is a significant distinction made between the sensation of pain and its perception. The sensation of pain refers to the nervous system response to noxious stimuli—this is also referred to as nociception (Kandel, Schwartz & Jessell 2000)--, whereas the perception of pain involves the conscious experience of the unpleasantness and intensity of being in pain (Sternberg 2007). There is dissociation between the two because an individual’s nervous system can sense noxious stimuli, but the individual may or may not have the subjective experience of being in pain. For the purposes of this study, the pain experience of the research subjects will be assessed through their observable pain behaviors. The nervous system has specialized peripheral neurons called nociceptors that allow individuals to detect noxious stimuli. These neurons are of two types: C-fibers and Aδ-fibers. Cfibers are unmyelinated and are sensitive to thermal (extreme hot and cold temperatures), mechanical (intense pressure), and chemical stimuli, and therefore are polymodal (Kandel et al. 2006). Aδ-fibers are thinly myelinated and respond to thermal and mechanical stimuli. Both of these fibers have the slowest conductance of all nervous system neurons because they are very small in diameter and have little or no myelination, two factors which are directly related to speed of signal conduction. (Sternberg 2007). There are also nonnociceptive fibers, Aβ-fibers, and Aα-fibers, which have larger Hippocampal Neurogenesis and Pain Behavior 6 diameters and are typically involved in the perception of innocuous stimuli, but can also have a role in modifying the perception of pain, which will be explained later in this section (Kandel et al. 2000). Nociceptors are primary sensory neurons that have their end terminals at the skin, joints, skeletal muscles, blood vessel walls and connective tissues, and have their cell bodies in the dorsal root ganglion (DRG). From the DRG, the fibers connect to the superficial layers of the dorsal horn of the spinal cord (Sternberg 2007). The position in the dorsal horn, or lamina, at which the nociceptor terminates depends on the type of fiber. The neurons that subsequently receive the signal from nociceptor afferents are known as pain transmission neurons (PTNs). PTNs have their cell bodies in the dorsal horn and carry the signal up to the brain. The signal is transmitted to the brain via five ascending pathways: the spinothalamic, spinoreticular, spinomesencephalic, cervicothalamic, and spinohypothalamic tracts. These pathways are distinct because they start in different laminae and terminate at different regions in the brain. The pathways are also involved in creating different aspects of the experience of pain, such as the affective, neuroendocrine, and cardiovascular responses. Consequently, the experience of pain is produced in the higher order areas of the cortex, such as the parietal and frontal lobe, and limbic system. The activation of this signal pathway occurs when noxious stimuli directly impinge on the peripheral extensions of these fibers. Oftentimes, both Aδ and C fibers are activated by noxious stimuli; the Aδ fibers produce a faster sensation of pain because they are myelinated, and are followed by the more aching pain caused by C fiber activation (Sternberg 2007). The mechanism whereby these stimuli depolarize the nociceptors is still under investigation; however, it is believed that the membranes of the nociceptor endings contain proteins that convert the mechanical, thermal, or chemical energy of the stimuli into electrical potential (Kandel et al. 2000). Once these neurons are depolarized, the signal is transmitted to the central nervous system by the release of the neurotransmitter glutamate in the spinal cord, as well as through the action of neuropeptides. Both of these chemicals work together to regulate nociceptive responses; glutamate activates AMPA Hippocampal Neurogenesis and Pain Behavior 7 (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors and causes an excitatory response, whereas neuropeptides enhance and prolong the action of glutamate. Pain measurement In this study, we will measure two types of pain through two experimental manipulations. We will measure tonic pain with the formalin test, as well as acute pain thresholds with the hotplate and tail withdrawal tests. The formalin test is used to measure animal responses to more continuous, moderate pain. As compared to other traditional pain tests that use thermal and mechanical stimuli, it is not testing acute pain, but rather pain involved with tissue injury. Therefore, it is believed that results from studies using the formalin test provide a more valid model for clinical pain (Dubuisson and Dennis 1977). The test involves a subcutaneous injection of formalin, an aqueous solution of formaldehyde, into the paw of the rodent. The hind paw is typically used, and will be used in the present study, because rodents do not usually lick this paw during normal grooming (Tjølsen, Berge, Hunskaar, Rosland, & Hole 1992). Therefore, observed licking of the hind paw is more likely to be a nociceptive response to the injection. There are two phases in the response to formalin, early and late. The early phase usually lasts five to ten minutes and is associated more directly to the activity of afferent C fibers (Heapy et al. 1987 as cited by Tjølsen et al. 1992). The early phase is followed by a quiescent period of about ten minutes. After the quiescent phase, the late response lasts from 20-40 minutes. The late phase appears to involve synaptic plasticity in the dorsal horn of the spinal cord that results from the initial barrage of C fiber activity and the inflammation response of peripheral tissues (Tjølsen et al. 1992). As a result of the strengthened response, animals must be euthanized shortly after testing. Otherwise, animals may experience long-lasting changes ranging from depilation and scarring to ulceration at the injection site (Rosland, Tjølsen, Mæhle, Hole 1900). Hippocampal Neurogenesis and Pain Behavior 8 We will also test the acute pain thresholds of the mice using a tail withdrawal test and a hotplate test, which are commonly used phasic pain tests. These tests are used to determine nociceptive thresholds, while the formalin test is a suprathreshold response (Wilson & Mogil 2001). This means that changing the intensity of the stimulus in the hotplate or tail withdrawal test will influence the latency of response, while changes in intensity of the formalin test will affect intensity and duration of response. The hotplate test involves placing the animal on a hotplate set to 53°C to measure a pain response to the acute thermal stimulus. The response that is typically measured is the hind paw fluttering or licking; hind paw lifting is often too subtle a response to accurately measure (Espejo & Mer, 1993). In this assay, front paw licking is also not measured, as it is a grooming response. Additionally, the mouse response to the hotplate test is stereotyped, and therefore easily measured (Bars, Gozariu, Cadden, 2001). However, the test is susceptible to effects of repetition (Gamble & Milne, 1989); therefore it will only be performed twice during the current study, with a period of 15 minutes in between each trial. In the tail withdrawal test, the distal portion of the mouse’s tail is submerged into a batch of circulating 49° water and the response time of withdrawing the tail is measured. At this temperature, response times are long enough to be accurately measured in the observer’s reaction time. It has been shown that this test is not affected by repeated exposures and measurements are reproducible (Tierny, Carmody, & Jamieson, 1991). Both hotplate and tail withdrawal tests are commonly used as a measure of animal nociception because they can be manipulated experimentally to examine factors that influence antinociception (Bannon & Malberg, 2007). It is noteworthy, however, that the behaviors measured in each test are mediated by different neural circuitry—as observed through studies using various techniques to modulate responses that have found conditions in which only one of the two tests are altered (Jensen & Yaksh, 1984; Fasmer, Berge, Tvieten, Hole, 1986). The hotplate test is supraspinally mediated, while the tail withdrawal test is spinally flexible (Wilson & Hippocampal Neurogenesis and Pain Behavior 9 Mogil, 2001). We will be using these tests to determine the effects of our manipulation on acute thermal pain. Pain Modulation The action of the PTNs can be modulated by various mechanisms, to both increase and decrease their effects. Both local nociceptive or non-nociceptive afferents and descending pathways from the brain can modulate the signal of the PTN. Other non-nociceptive afferents can affect nociception because they sometimes terminate at the same lamina as the nociceptors and can have an excitatory or inhibitory effect. In general, the non-nociceptive afferents inhibit the ascending signal to the brain. This idea was postulated as the “gate control theory” in 1965 by Mezlack and Wall. Descending pathways have modulatory affects on pain because they too can inhibit the firing of PTNs in the laminae of the dorsal horn, thereby producing an analgesic affect. Evidence for this pathway was found through electrical stimulation of midbrain sites (Reynolds 1969; Mayer et al 1971). The action of the descending pathways can be either directly or indirectly inhibitory, or can interact with endogenous opioid-containing pathways (Kandel et al. 2000). The body produces endogenous opioids, such as enkephalins, β-endorphin, and dynorphins, which have analgesic effects by inhibiting the firing of PTNs. Opioids have effects on PTNs through the action of the numerous opioid receptors present in the dorsal horn of the spinal cord. This inhibition can occur through postsynaptic inhibition or by presynaptic inhibition of glutamate release (Basbaum et al. 2000). Another form of inhibition of nociceptive pathways resulting in analgesia is stress-induced analgesia (SIA). This form of analgesia occurs in animals during stressful conditions. It suppresses the typical responses to pain—such as reflex, withdrawal, and rest and recuperation (Kandel et al. 2000)—which could be disadvantageous in stressful situations. The mechanism of SIA will be explained further in a later section. Hippocampal Neurogenesis and Pain Behavior 10 On the other hand, nociception can be increased by various mechanisms. Hyperalgesia, an increased response to noxious stimuli (Ji Kohno, Moore, & Woolf 2003), has been widely researched and can be caused by sensitization, both peripherally and centrally. Peripheral sensitization refers to changes in the peripheral terminals of nociceptors, while central sensitization is due to changes occurring in the dorsal horn of the spinal cord. During an injury, there is a release of various chemicals from dead and dying cells in the region of tissue damage. These chemicals increase the sensitivity and excitability of the nociceptors in the periphery by activating signaling pathways that change the threshold and kinetics of receptors and ion channels. Therefore, there is increased pain sensitivity in the region of inflammation (Ji et al. 2003). Central sensitization refers to synaptic strengthening that occurs in the synapses of the dorsal horn of the spinal cord as a result of intense peripheral tissue damage caused by a noxious stimulus. The damage causes the nociceptors to fire at a great rate, strengthening the connection between the nociceptors and PTNs. Thus, damage causes increased sensitivity after the injury, and allows for PTNs to be activated by lower intensity stimuli and lower threshold primary afferents, which are usually activated by innocuous stimuli (Sternberg 2007). This sensation of pain caused by innocuous stimuli is called allodynia. Although the perception of increased pain resulting from central sensitization feels as if it originates in the periphery, the heightened perception is actually the result of strengthened connectivity in the central nervous system. This phenomenon is unlike peripheral sensitization, which results from changes in receptors and ion channels specifically in the periphery. The strengthening that results from central sensitization is a form of synaptic plasticity. Long-term potentiation (LTP), another widely studied form of synaptic plasticity, has been compared to central sensitization because they share common mechanisms. LTP and its relevance to central sensitization will be further explained in the following section.