<p>Impulses coming from peripheries are modified in the spinal cord and transmitted to the brain. Several neurotransmitters have been involved in the processing of impulses in the spinal dorsal horn. Acetylcholine (ACh) is one of many neurotransmitters involved in the regulation of nociception in the spinal cord. In this study we investigated the role of nicotinic, muscarinic, serotonergic and GABA receptors in the regulation of spinal ACh release since these receptors are reported to be involved in spinal nociceptive processes.</p><p>Different receptor ligands were infused intraspinally via microdialysis and the spinal ACh release was measured by on-line HPLC. Receptor-ligand binding studies were performed with spinal cord homogenates as well as receptors expressed in cells.</p><p>In the first study, we found that nicotine and some of the nicotinic antagonists used increased ACh release suggesting that spinal ACh release is regulated by different nAChRs. Nicotine and nicotinic agonists may act on different types of receptors with different affinity to produce the observed net effect of increased ACh release. We propose the possibility of an involvement of three different nicotinic receptor subtypes in the regulation of spinal ACh release. </p><p>The effect of epibatidine, which is regarded as a nicotinic agonist, on muscarinic receptors was investigated in the second study. We propose that epibatidine, in μM concentrations, is a partial muscarinic receptor agonist that may interact with spinal muscarinic receptors to increase ACh release. The dual action on both nAChRs and mAChRs may explain the potent analgesic effect observed after intra-spinal epibatidine administration.</p><p>In the third study, we investigated the role of serotonin receptor involvement in ACh release control. The results suggest that only 5-HT_1A and 5-HT_2A receptors are involved in spinal ACh release. Considering current knowledge, the most probable location of 5-HT_2A receptors is on cholinergic neurones. On activation of the 5-HT_2A receptors the cellular excitability of cholinergic neurones is increased which results in an increasing ACh release. The 5-HT_1A receptors might be located on cell bodies of GABA neurones which inhibit the firing rate of the GABA neurones when activated by serotonin. </p><p>In the fourth study, we investigated the GABA receptor involvement in the regulation in spinal ACh release. We found that GABA_A receptors are tonically inhibiting spinal ACh release. The results further suggest that GABA_B receptors also are involved in the regulation of spinal ACh release. However, unlike GABA_A antagonists, GABA_B antagonists do not increase ACh release. This suggests that GABA_B receptors are not tonically regulating the spinal ACh release. </p>

During repetitive stimulation of motor nerve terminals, mitochondrial Ca2+ uptake limits increases in free cytosolic [Ca2+] and helps ensure faithful neuromuscular transmission. Changes in cytosolic [Ca2+] and in mitochondrial [Ca2+] as well as changes in mitochondrial membrane potential (Psi m) were studied in mouse motor nerve terminals using Ca2+ sensitive indicator and potentiometric dyes, respectively. Trains of action potentials (APs) at 50 to 100 Hz produced a rapid increase in mitochondrial [Ca2+] followed by a plateau which usually continued beyond the end of stimulation. After stimulation, mitochondrial [Ca2+] decayed back to baseline over the course of tens of seconds to minutes. Increasing the Ca2+ load delivered to the terminal by increasing the number of stimuli (500-2000), increasing bath [Ca2+], or prolonging the AP with 3,4-diaminopyridine (3-4, DAP, 100 micromolar), prolonged the post-stimulation decay of mitochondrial [Ca2+] without increasing the amplitude of the plateau. Inhibiting openings of the mitochondrial permeability transition pore with cyclosporin A (5 micromolar) had no significant effect on the decay of mitochondrial [Ca2+]. Inhibition of the mitochondrial Na+-Ca2+ exchanger with CGP-37157 (50 micromolar) dramatically prolonged the post-stimulation decay of mitochondrial [Ca2+], reduced post-stimulation residual cytosolic [Ca2+], and reduced the amplitude of end-plate potentials evoked after the end of stimulation. Stimulation-induced mitochondrial Ca2+ uptake resulted in Psi m depolarizations that were small or undetectable at near-physiological temperatures (~30 degrees C). Their amplitude became larger at lower temperatures (~20 degrees C), or when AP duration was increased with 3,4-DAP (20 micromolar). Psi m depolarizations were inhibited by lowering bath [Ca2+] or by blocking P/Q-type Ca2+ channels with omega-agatoxin (0.3 micromolar). Partial inhibition of complex I of the electron transport chain (ETC) with rotenone (50 nM) increased the amplitude of stimulation-induced Psi m depolarizations. These findings suggest that: (1) Ca2+ extrusion from motor terminal mitochondria occurs primarily via the Na+-Ca2+ exchanger and helps sustain post-tetanic transmitter release, and (2) that the depolarization of Psi m that accompanies Ca2+ uptake is limited by accelerated proton extrusion via the ETC.

Impulses coming from peripheries are modified in the spinal cord and transmitted to the brain. Several neurotransmitters have been involved in the processing of impulses in the spinal dorsal horn. Acetylcholine (ACh) is one of many neurotransmitters involved in the regulation of nociception in the spinal cord. In this study we investigated the role of nicotinic, muscarinic, serotonergic and GABA receptors in the regulation of spinal ACh release since these receptors are reported to be involved in spinal nociceptive processes. Different receptor ligands were infused intraspinally via microdialysis and the spinal ACh release was measured by on-line HPLC. Receptor-ligand binding studies were performed with spinal cord homogenates as well as receptors expressed in cells. In the first study, we found that nicotine and some of the nicotinic antagonists used increased ACh release suggesting that spinal ACh release is regulated by different nAChRs. Nicotine and nicotinic agonists may act on different types of receptors with different affinity to produce the observed net effect of increased ACh release. We propose the possibility of an involvement of three different nicotinic receptor subtypes in the regulation of spinal ACh release. The effect of epibatidine, which is regarded as a nicotinic agonist, on muscarinic receptors was investigated in the second study. We propose that epibatidine, in μM concentrations, is a partial muscarinic receptor agonist that may interact with spinal muscarinic receptors to increase ACh release. The dual action on both nAChRs and mAChRs may explain the potent analgesic effect observed after intra-spinal epibatidine administration. In the third study, we investigated the role of serotonin receptor involvement in ACh release control. The results suggest that only 5-HT1A and 5-HT2A receptors are involved in spinal ACh release. Considering current knowledge, the most probable location of 5-HT2A receptors is on cholinergic neurones. On activation of the 5-HT2A receptors the cellular excitability of cholinergic neurones is increased which results in an increasing ACh release. The 5-HT1A receptors might be located on cell bodies of GABA neurones which inhibit the firing rate of the GABA neurones when activated by serotonin. In the fourth study, we investigated the GABA receptor involvement in the regulation in spinal ACh release. We found that GABAA receptors are tonically inhibiting spinal ACh release. The results further suggest that GABAB receptors also are involved in the regulation of spinal ACh release. However, unlike GABAA antagonists, GABAB antagonists do not increase ACh release. This suggests that GABAB receptors are not tonically regulating the spinal ACh release.

Assessment of the microcirculation is of major importance in understanding the physiology of the vasculature and in assessing te vascular effects of pathological conditions such as diabetes, hypertension and sepsis. Transdermal iontophoresis can be used to non‐invasively introduce vasoactive drugs into the skin. The response to these drugs of the local cutaneous microvasculature can be measured by laser Doppler flowmetry methods. Although these techniques have been used together for over two decades, there are still important methodological issues to be resolved. This work is aimed at optimizing transdermal iontophoresis as a tool for microvascular assessment by focusing on the main methdological issues: non‐specific vasodilatation, drug delivery protocols and analysis of blood flow data. Non‐specific vasodilatation, an increase blood flow during iontophoresis of non‐vasoactive compounds, is an important problem as it interferes with the response to the administered drug. By investigating this effect in healthy volunteers, we found that the extent of the non‐specific response differs between the positive and negative electrode and that it is dependent on the voltage over the skin andon the ionic strength of the vehicle in which the drug is dissolved. We also found that the extent of the non‐specific response could be reduced by applying local anesthetics and by pre‐treatment with antihistamine drugs. These results suggest that non‐specific effects could be mediated by depolarization or hyperpolarisation of cells, triggering neural and histamine related mechanisms that finally lead to vasodilatation of the local microvasculature. To prevent non‐specific effects from occurring during the experiments, our results show that the current strength and the total electric charge during iontophoresis should be limited to 0.02 mA and12 mC, respectively. Furthermore, drug solutions at physiological ionic strengths should be used. Under these conditions, adequate responses to the most commonly used drugs, acetylcholine (ACh) and sodium nitroprusside (SNP), are obtained while no significant non‐specific vasodilatation occurs. The results of our investigations show that blood responses to ACh and SNP applied by a single iontophoretic pulse can well be escribed by conventional dose‐response models, which enables a more powerful analysis and comparison between drugs or possibly patient groups as compared with conventional aalysis methods. Finally, we have incorporated drug transport and physiological response to the local drug concentration during iontophoresis of vasoactve drugs into a single model. Validation of this model using measured responses to ACh and SNP shows that the commonly used assumption that the local drug concentration during iontophoresis is linearly proportional to the electric charge may not be valid. / Mikrocirkulationen, som inbegriper kroppens minsta blodkärl, transporterar syre och näringsämnen till våra celler. Vissa sjukdomar, som diabetes, hjärt‐kärlsjukdom och akut blodförgiftning leder till förändringar hos mikrocirkulationen. Mekanismerna bakom dessa förändringar är delvis okända. Det finns därför ett stort behov av kliniska mättekniker som kan bedöma mikrocirkulationens funktion. Vid jontofores placeras en elektrod tillsammans med ett läkemedel på huden. När en svag elektrisk ström anbringas transporteras läkemedlet ner genom hudlagren. Effekterna av ett kärlaktivt läkemedel som appliceras på detta sätt kan sedan avläsas non‐invasivt med laser Doppler‐teknik. En stor fördel med jontoforesmetoden, förutom att den är non‐invasiv, är att läkemedelsdoserna som tillförs kroppen är mycket små och därmed ger de inte upphov till några systemiska bi‐effekter. I avhandlingen presenteras forskning, vilkas målsättning är att lösa några av de viktiga frågorna kring transdermal jontofores så att tekniken optimeras för att denskall kunna brukas som ett verktyg vid kliniska undersökningar av mikrocirkulationen. Den första delen ägnas ett fenomen som kallas ospecifik vasodilatation. Det uppstår vid jontofores av substanser som är inte kärlaktiv, som vatten och koksaltlösning. Resultaten från dessa försök indikerar att den ospecifika vasodilatationen beror på framför allt spänningen över huden, vilken i sin tur är relaterad till jon‐koncentrationen hos läkemedelslösningen. Vidare registreras att mekanismen bakom den ospecifika vasodilatationen delvis är neuralt medierad genom att de till stor del år att förhindra med hjälp av lokal bedövning. Dessutom leder förbehandling med anti‐histamina läkemedel till minskade ospecifika reaktioner, vilket också indikerar att lokala inflammatoriska processer är inblandande. Den andra delen av avhandlingen ägnas att optimera försöksprotokollen för jontofores. Till att börja med utvecklas ett protokoll som ger ett adekvat läkemedelssvar samtidigt som ospecifika effekter minimeras. Det visar sig är möjligt genom att begränsa strömstyrkan och den elektriska laddningen under jontoforesen och genom att använd läkemedelslösningar som har en fysiologisk jonstyrka. Resultaten visar också att blodflödesförändringen som registreras under jontofores av acetylkolin och natriumnitroprussid kan eskrivas med hjälp av konventionella dos‐responsmodeller, vilket möjliggör en mer exakt analys av det mikrocirkulatoriska svaret samt underlättar jämförelse mellan olika läkemedel elle patientgrupper. Slutligen presenteras en mekanistisk model för det mikrocirkulatoriska svaret vid jontofores. Modellen beskriver läkemedlets transport från elektroden ner genom huden, clearance i huden vilken beror på diffusion och det lokala blodflödet, samt förändringen i blodflöde som sker på grund av läkemedlet. Modellen valideras genom försök på försökspersoner och resultaten visar att förändringarna i blodflödet åstadkommet av acetylklin och natriumnitroprussid med denna modell kan beskrivas på ett exakt sätt. Vidare visar resultaten att det sker en betydande clearance av läkemedel i huden under jontofores. Detta har väsentlig betydelse när man ska uppskatta den lokala jontoforesdosen. / The author changed surname from Droog to Tesselaar in January 2006.

The spinal cord is an important component in the processing and modulation of painful stimuli. Nerve signals from the periphery are relayed and further conducted to the brain (nociception) in the spinal cord, and the most essential modulation of painful information (antinociception) occurs here. Several neurotransmitters are involved in spinal pain modulation, among them acetylcholine. However, the role of acetylcholine has previously been little investigated. In the present thesis, the acetylcholine release in the spinal cord was studied in vivo. By using spinal microdialysis on anaesthetised rats, the effects on the intraspinal acetylcholine release of various receptor ligands and analgesic agents were examined. This, together with pain behavioural tests and in vitro pharmacological assays, was used to evaluate the role of acetylcholine in spinal pain modulation. The four studies in this thesis resulted in the following conclusions: An increased release of spinal acetylcholine is associated with an elevated pain threshold, while a decreased acetylcholine release is associated with hyperalgesia, as seen after systemic treatment with a muscarinic agonist and an antagonist. Lidocaine is a potent analgesic when given systemically. It was found to produce an increase of intraspinal acetylcholine after intravenous injection of analgesic doses. This effect was attenuated after muscarinic, and abolished after nicotinic, receptor blockade. Various a2-adrenergic ligands, associated with nociceptive or antinociceptive effects, were found to affect intraspinal acetylcholine release via action on nicotinic receptors. Finally, the involvement of spinal acetylcholine in the analgesic effects of aspirin and paracetamol was examined. It was found that spinal acetylcholine could participate in the analgesic effects of aspirin, but not of paracetamol. The present thesis provides data that clearly demonstrate a relationship between intraspinal acetylcholine and antinociception, and elucidate interactions between acetylcholine and other mechanisms that mediate antinociception in the spinal cord.

Tendinosis is a condition of chronic pain that afflicts several human tendons, not least the patellar tendon, in which case it is often clinically referred to as ‘jumper’s knee’. The exact mechanisms behind tendinosis are yet not fully understood. One draw-back in the case of patellar tendinosis has been the lack of knowledge of the innervation patterns of the human patellar tendon. It cannot be excluded that the processes of tendinosis are influenced by nerve mediators, released from nerve endings or from stimulated cells inside the tendon. Thus, the studies of the present thesis aimed to 1) map the general, sensory, cholinergic and sympathetic innervation patterns of the human patellar tendon, in both the tendon tissue proper and the loose paratendinous connective tissue surrounding the tendon, and 2) investigate the possible existence of a production of signal substances, traditionally associated with neurons, in non-neuronal tendon cells, and to see if there are signs of local cholinergic and catecholaminergic signaling pathways. Biopsies of both normal pain-free patellar tendons and patellar tendons from patients with chronic painful tendinosis were collected and investigated. The main method utilized was immunohistochemistry, using antibodies directed against synthesizing enzymes for acetylcholine and catecholamines, against muscarinic and adrenergic receptors, and against markers of general and sensory innervation. In situ hybridization (ISH) to detect mRNA for the cholinergic/catecholaminergic synthesizing enzymes was also used. It was found that the loose paratendinous connective tissue of the patellar tendon was rather richly innervated by nerve structures. These consisted of large nerve fascicles, as well as perivascular innervation in the walls of some of the larger arteries and smaller blood vessels. It was found that part of the nerve structures corresponded to sensory afferents, and that some conformed to cholinergic and, especially, sympathetic nerve fibers. The tendon tissue proper was strikingly less innervated than the paratendinous tissue. The sparse innervation that was found in the tendon tissue proper was seen in narrow zones of loose connective tissue and blood vessels, interspersed between the collagen bundles. The overall impression was that the patterns of distribution of the general, sensory, and autonomic innervations of tendinosis tendon tissue were similar to those of normal tendon tissue proper. The most pioneering findings were the immunohistochemical observations of an expression of enzymes related to production of both acetylcholine and catecholamines within the tendon cells (tenocytes) themselves, as well as of a presence of the receptors for these substances on the same cells; features that were predominantly seen in tendinosis tendons. The observations of the synthesizing enzymes for acetylcholine and catecholamines in tenocytes were confirmed by ISH findings of mRNA for these enzymes in the tenocytes. Immunoreactions for muscarinic and adrenergic receptors were also found in blood vessel walls and in some of the nerve fascicles. In summary, this thesis presents novel information on the innervation patterns of the human patellar tendon, in healthy individuals with pain-free tendons as well as in patients with chronic painful tendinosis. Furthermore, it gives the first evidence of the presence of a local, non-neuronal production in the tendon tissue of signal substances normally seen in neurons, and a basis for these substances to affect the tenocytes as these cells also display muscarinic and adrenergic receptors. Thus, the results indicate an existence of autocrine and/or paracrine cholinergic/catecholaminergic systems in the tendon tissue; systems that seem to be up-regulated in tendinosis. This is of great interest as it is known that stimulation of receptors for both catecholamines and acetylcholine can lead to cell proliferation, interfere with pain sensation, influence collagen production, and take part in vasoregulation, as well as, in the case of adrenergic receptors, promote cell degeneration and apotosis. All these processes represent biological functions/events that are reported to be affected in tendinosis. In conclusion, despite the fact that there is very limited innervation within the patellar tendon tissue proper, it is here shown that effects of signal substances traditionally associated with neurons seem to occur in the tissue, via a local production of these substances in tenocytes.

The presented studies were performed to investigate the effects of motion sickness (MS) on human autonomic and behavioural thermoregulatory mechanisms during cold stress and in a thermoneutral environment. The roles of histaminergic and cholinergic neuron systems in autonomic thermoregulation and MS-dependent dysfunction of autonomic thermoregulation were studied using a histamine-receptor blocker, dimenhydrinate (DMH), and a muscarine-receptor blocker, scopolamine (Scop). In addition, the effects of these substances on MS-induced nausea and perceptual thermoregulatory responses were studied. MS was found to lower core temperature, during cold stress by attenuation of cold-induced vasoconstriction and decreased shivering thermogenesis, and in a thermoneutral environment by inducing sweating and vasodilatation. The increased core cooling during cold stress was counteracted by DMH but not by Scop. In a thermoneutral environment, the temperature was perceived as uncomfortably warm during and after the MS provocation despite decreases in both core and skin temperature. No such effect was seen during cold-water immersion. Both pharmacologic substances had per se different effects on autonomic thermoregulatory responses during cold stress. Scop decreased heat preservation, but did not affect core cooling, while DMH reduced the rate of core cooling through increased shivering thermogenesis. Both DMH and Scop per se decreased thermal discomfort during cold-water immersion.Findings support the notion of modulating roles of histamine (H) and acetylcholine (Ach) in autonomic thermoregulation and during MS. MS activates cholinergic and histaminergic pathways, thereby increasing the levels of H and Ach in several neuro-anatomical structures. As a secondary effect, MS also elevates blood levels of several neuropeptides, which in turn would influence central and/or peripheral thermoregulatory responses.In conclusion, MS may predispose to hypothermia, by impairment of autonomic thermoregulation in both cold and thermoneutral environments and by modulation of behavioural thermoregulatory input signals. This might have significant implications for survival in maritime accidents. / <p>Medicine doktorsexamen</p>

Feature binding refers to the fundamental challenge of the brain to integrate sensory information registered by distinct brain regions to form a unified neural representation of a stimulus. While the human cognitive literature has established that attentional processes in a frontoparietal cortical network support feature binding, the neurochemical contributions to this attentional process remain unknown. Using systemic administration of the cholinergic muscarinic receptor antagonist scopolamine and a digging-based rat feature binding task that used both odor and texture stimuli, it was demonstrated that blockade of acetylcholine (ACh) at the muscarinic receptors impaired rats’ ability to feature bind at encoding, and it was proposed that ACh may support the attentional processes necessary for feature binding (Botly & De Rosa, 2007). This series of experiments further investigated a role for ACh and the cholinergic basal forebrain (BF) in feature binding. In Experiment 1, a cross-species experimental design was employed in which rats under the systemic influence of scopolamine and human participants under divided-attention performed comparable feature binding tasks using odor stimuli for rats and coloured-shape visual stimuli for humans. Given the comparable performance impairments demonstrated by both species, Experiment 1 suggested that ACh acting at muscarinic receptors supports the attentional processes necessary for feature binding at encoding. Experiments 2-4 investigated the functional neuroanatomy of feature binding using bilateral quisqualic acid excitotoxic (Experiment 2) and 192 IgG-saporin cholinergic immunotoxic (Experiments 3 and 4) brain lesions that were assessed for completeness using histological and immunohistological analyses. Using the crossmodal digging-based rat feature binding task, Experiment 2 revealed that the nucleus basalis magnocellularis (NBM) of the BF is critically involved in feature binding, and Experiment 3 revealed that cholinergic neurons in the NBM are necessary for feature binding at encoding. Lastly, in Experiment 4, rats performed visual search, the standard test of feature binding in humans, with touchscreen-equipped operant chambers. Here it was also revealed that cholinergic neurons in the NBM of the BF are critical for efficient visual search. Taken together, these behavioural, pharmacological, and brain-lesion findings have provided insights into the neurochemical contributions to the fundamental attentional process of feature binding.

Feature binding refers to the fundamental challenge of the brain to integrate sensory information registered by distinct brain regions to form a unified neural representation of a stimulus. While the human cognitive literature has established that attentional processes in a frontoparietal cortical network support feature binding, the neurochemical contributions to this attentional process remain unknown. Using systemic administration of the cholinergic muscarinic receptor antagonist scopolamine and a digging-based rat feature binding task that used both odor and texture stimuli, it was demonstrated that blockade of acetylcholine (ACh) at the muscarinic receptors impaired rats’ ability to feature bind at encoding, and it was proposed that ACh may support the attentional processes necessary for feature binding (Botly & De Rosa, 2007). This series of experiments further investigated a role for ACh and the cholinergic basal forebrain (BF) in feature binding. In Experiment 1, a cross-species experimental design was employed in which rats under the systemic influence of scopolamine and human participants under divided-attention performed comparable feature binding tasks using odor stimuli for rats and coloured-shape visual stimuli for humans. Given the comparable performance impairments demonstrated by both species, Experiment 1 suggested that ACh acting at muscarinic receptors supports the attentional processes necessary for feature binding at encoding. Experiments 2-4 investigated the functional neuroanatomy of feature binding using bilateral quisqualic acid excitotoxic (Experiment 2) and 192 IgG-saporin cholinergic immunotoxic (Experiments 3 and 4) brain lesions that were assessed for completeness using histological and immunohistological analyses. Using the crossmodal digging-based rat feature binding task, Experiment 2 revealed that the nucleus basalis magnocellularis (NBM) of the BF is critically involved in feature binding, and Experiment 3 revealed that cholinergic neurons in the NBM are necessary for feature binding at encoding. Lastly, in Experiment 4, rats performed visual search, the standard test of feature binding in humans, with touchscreen-equipped operant chambers. Here it was also revealed that cholinergic neurons in the NBM of the BF are critical for efficient visual search. Taken together, these behavioural, pharmacological, and brain-lesion findings have provided insights into the neurochemical contributions to the fundamental attentional process of feature binding.

Extensive evidence suggests that glucose has both positive and negative effects on memory and these effects likely involve an influence on the brain. For instance, direct infusions of glucose into the septum (MS) or hippocampus can enhance or impair memory. The present set of experiments attempted to determine the different conditions that dissociate the memory-enhancing and -impairing effects of glucose in rats. Specifically, these experiments examined the effects of glucose in spontaneous alternation, a measure of spatial working memory and shock avoidance, an index of emontional long-term memory. The results showed that the memory-impairing effects of MS infusions of glucose are not concentration-dependent. These data also indicated that the memory-impairing effects of MS glucose elevations are specific to gamma-aminobutyric acid GABA receptor activation but do not depend on increases in MS GABA synthesis or release. Importantly, we showed that the memory-impairing interaction between MS glucose and GABA agonists does not generalize to the hippocampus, suggesting the memory-modulating effects of glucose are brain region-dependent. We showed further that these brain region-dependent effects of glucose are not due to difference in basal extracellular glucose levels. Moreover, these findings showed that the memory-enhancing effects of hippocampus glucose override the memory-impairing interaction between MS glucose and GABA. These findings are important because they are the first to show that the memory-modulating effects of glucose are both neurotransmitter- and brain region-dependent. Furthermore, these findings provide preliminary evidence suggesting that the memory-impairing effects of MS glucose may involve compromised hippocampal function. These data also suggest the memory-impairing effects of MS co-infusions of glucose with GABA agonists likely involve an influence on the GABAergic SH projection. Finally, these findings demonstrate the mnemonic and neurochemical consequences of glucose in the MS and hippocampus, two brain regions affected by normal aging, Alzheimer’s disease, and diabetes.