In this thesis, a major focus has been to examine the possible mechanism (s) mediating the neuro-excitatory behavioural effects as well as the "anti-analgesic" actions of morphine-3- glucuronide (M3G), the major metabolite of morphine in humans and rodents. M3G has been implicated in the production of the neuro-excitatory side-effects (e.g. allodynia, myoclonus and seizures) seen in some patients receiving chronic high-dose systemic morphine for cancer pain management. As morphine remains the drug of choice for the treatment of moderate to severe cancer pain, understanding the mechanism through which M3G evokes its neuro-excitatory behavioural effects and/or its "anti-analgesic" actions, is clearly desirable. Once the mechanism(s) underlying M3G's neuro-excitatory behavioural effects is understood, it may be possible to improve morphine dosing regimens to prevent the development of opioid-induced neuro-excitatory behavioural side-effects, whilst maintaining satisfactory pain relief in cancer patients. To address this issue, I have used a range of in vitro and in vivo experimental approaches including calcium imaging techniques and neurotoxicity assays in cultured neurones, together with intracerebroventricular (i.c.v.) and intrathecal (i.t.) drug dosing for behavioural studies in rats, respectively.
In chapter 1,1 have reviewed the pharmacological actions of morphine and M3G, as well as the possible factors contributing to neuro-excitation evoked by these compounds. Additionally, a brief discussion of the applicability and/or the strengths and weaknesses of several of the experimental techniques used in my doctoral studies has also been included, for example, the developmental changes that take place in cultured neurones during the culture period, choice of the appropriate fluorescent calcium indicator for the measurement of cytosolic free calcium concentrations in cultured neurones, and the in vitro methods available for the assessment of neurotoxicity.
In chapter 2,1 described the development of a model system comprising primary cultures of embryonic rat hippocampal neurones in conjunction with state-of-the-art fluorescence digital imaging techniques to investigate whether M3G could enhance spontaneous glutamate-induced Ca2+ oscillations in cultured rat hippocampal neurones. Fluo-3 fluorescence digital imaging techniques were used to qualitatively assess the acute effects of M3G on [Ca2+]CYT in these cultured neurones. My studies demonstrated that cultured hippocampal neurones were synaptically active and dependent primarily on glutamatergic synaptic transmission, consistent with previous reports in the literature. Moreover, M3G was shown to consistently increase [Ca2+]CYT via a predominantly non-opioid mechanism, thereby providing further indirect evidence that glutamatergic neurotransmission may be involved in mediating M3G's neuro-excitatory behavioural side-effects.
In chapter 3,I confirmed my findings described in chapter 2 showing that M3G may mediate its neuro-excitatory effects via the enhancement of glutamatergic neurotransmission, and I undertook a series of additional experiments to further examine this issue. Specifically, cultured embryonic rat hippocampal neurones were exposed to a range of compounds including M3G, dynorphin A (2-17), dynorphin-antiserum, various glutamate agonists (e.g. glutamate, NMDA and AMPA) and antagonists (e.g. LY274614, MK-801 and CNQX), ion channel blockers (e.g. calcium channel blockers; the sodium channel blocker, TTX, and the potassium channel blocker, 4-AP) as well as the GABAB receptor agonist, baclofen, and the GABAA receptor agonist, midazolam. My results show that M3G indirectly activates the NMDA receptor via a predominantly non-opioidergic mechanism. Moreover, M3G appears to produce its neuro-excitatory effects predominantly via indirect modulation of pre-synaptic neurotransmitter release, which in turn indirectly activates the NMDA receptor.
In chapter 4, the degree of neurotoxicity evoked by both acute and chronic exposure of cultured neurones to M3G, was investigated using imaging techniques in a manner similar to that described in chapter 3 and a lactase dehydrogenase (LDH) assay, respectively. My results showed that although acute exposure of neurones to M3G (50 μM) produced a large increase in [Ca2+]CYT, this increase was relatively transient when compared with glutamate (500 μM), leading to the proposal that M3G may not be neurotoxic. Additionally, prolonged exposure (24 h) of primary cultures of hippocampal and cerebellar granular neurones to each of M3G (5 and 50 µM) and morphine (5 and 50 μM), showed that when assessed using the LDH assay, the degree of neurotoxicity (if any) induced by high-dose M3G (50 μM) and high-dose morphine (50 μM) was significantly less (P < 0.0001) than that elicited by the well established excitotoxin, L-glutamate. Collectively, these findings suggest that M3G is unlikely to be neurotoxic in vivo.
The aim of my study described in chapter 5 was to develop a protocol using neurones cultured in microplate format, together with the NOVOstar® fluorescence microplate reader, to design an assay that would be suitable for moderate to high-throughput initial screening of compounds that may modulate glutamatergic neurotransmission. I successfully achieved this aim. Additionally, the protocol that I developed may be of benefit in future studies to assess the quantitative effects of M3G (e.g. assessment of quantitative changes in [Ca2+ ]CYT), as it allows a number of experiments to be conducted in a short period of time, when compared with conventional high resolution fluorescence microscopy.
As a number of recent studies have reported that intrathecal administration of Dyn A and its fragment peptides produce a similar profile of behavioural excitation (including allodynia and hyperalgesia) to that produced by centrally administered M3G or high-dose morphine, in rodents (Vanderah et al, 1996; Laughlin et al, 1997; Vanderah et al, 2000), my studies described in chapter 6 were designed to determine whether the neuro-excitatory behavioural effects evoked by i.c.v. and i.t. M3G in rats, could be attenuated by pre-treatment of rats with i.c.v. and/or i.t. dynorphin-antiserum prior to M3G administration. Although pre-treatment of rats with dynorphin-antiserum did not completely block either i.c.v. or i.t. M3G's neuroexcitatory behavioural effects, dynorphin-antiserum pre-treatment appeared to attenuate the severity of i.c.v. M3G (11 nmol)-evoked tonic-clonic convulsions. Moreover, administration of dynorphin-antiserum prior to i.t. M3G administration abolished M3G-induced explosive motor behaviour at 25 and 35 min post-dosing. Collectively, these findings suggest that dynorphin may have a role in the maintenance of some, but not all, of M3G-evoked neuro-excitatory behaviours. These results are consistent with my findings in chapter 3 showing that although pre-treatment of neurones with dynorphin-antiserum failed to prevent M3G-induced increases in [Ca2+ ]CYT, it may have attenuated M3G's effects, although further investigation is required to clarify this issue.
In my next series of experiments described in chapter 7, I re-examined the putative "antianalgesic" pharmacology of M3G in rats using the tail flick test, in order to gain insight into recent findings in our laboratory, whereby there were apparently large differences in the tail-flick latency results depending upon whether the noxious thermal stimulus was applied to the ventral or the dorsal surface of the rat's tail. In this study, I used three different tail-flick latency devices, viz (i) Apparatus I which allowed application of a noxious thermal stimulus to either the dorsal or the ventral surface of the rat's tail using the same heat source, in a sequential manner, (ii) the Columbus Instrument's Tail Flick Device which applied noxious thermal to the ventral aspect of the rat's tail, and (iii) the Warm water tail-flick method. My results showed that the apparent "anti-analgesic" or antinociceptive effects of bolus doses of i.c.v. M3G (4.0-6.5 nmol), administered alone or in combination with i.c.v. morphine (53-70 nmol), when assessed using the tail-flick test, appear to depend upon the exact dose of i.c.v. M3G administered, precise tail-flick test parameters utilized, including the surface area of the skin stimulated and whether the thermal stimulus is applied to the dorsal or the ventral surface of the rat's tail or to the entire most distal 2.5 cm of the rat's tail. My findings clearly show that a systematic re-examination of M3G's intrinsic pharmacology is required using a broad range of M3G doses, several routes of administration and multiple nociceptive tests.
As a first step towards a systematic re-examination of M3G's intrinsic pharmacology, my studies described in chapter 8, were designed to investigate whether i.c.v. M3G could modulate von-Frey paw withdrawal thresholds (PWTs) secondary to the application of a non-noxious stimulus of light pressure to the rat hindpaw in order to examine whether M3G evoked mechanical (tactile) allodynia. My results show that low-dose (0.1-2.0 nmol) i.c.v. M3G produces tactile allodynia, whereas higher doses (3.0-5.0 nmol) of i.c.v. M3G appear to produce anti-allodynic actions, although there were potentially confounding intermittent mild to moderate neuro-excitatory behavioural effects present in some of the animals that received these higher doses. My findings clearly show that i.c.v. M3G dose selection is critical, such that co-administration of i.c.v. morphine with relatively low doses of i.c.v. M3G (0.1-1.0 nmol) would be appropriate for the examination of the putative "anti-analgesic" effects of M3G, whereas higher doses of i.c.v. M3G (11 nmol) are more useful for investigating the modulation of M3G-evoked neuro-excitatory motor behaviours in rats.
In the final chapter of this thesis, I have drawn some general conclusions regarding the findings of my doctoral research and have made some suggestions with respect to possible future investigations.