Endomorphin 1 and 2 are two tetrapeptides recently isolated from bovine as well as human brains and proposed to be the endogenous ligand for the μ- opiate receptor Opioid compounds expressing μ-receptor preference are generally potent analgesics. The spinal cord dorsal horn is considered to be an important site for the processing of sensory information including pain. The discovery that endomorphins produced greater analgesia in mice upon intrathecal as compared to intracerebroventricular injections raises the possibility that dorsal horn neurons may represent the anatomic site upon which endomorphins exert their analgesic effects. We report here the detection of endomorphin 2-immunoreactive fiber-like elements in superficial layers of the rat dorsal horn by immunohistochemical techniques. Whole-cell patch recordings from substantia gelatinosa neurons of cervical spinal cord slices revealed two conspicuous effects of exogenously applied endomorphin 1 and 2: (i) depression of excitatory postsynaptic potentials evoked by stimulation of dorsal root entry zone, and (ii) hyperpolarization of substantia gelatinosa neurons. These effects were reversed by the selective μ-opiate receptor antagonist β-funaltrexamine. Collectively, the detection of endomorphin-like immunoreactivity in nerve fibers of the superficial layers and the inhibitory action of endomorphins on substantia gelatinosa neurons provide further support for a potential role of these two peptides in spinal nociception.

Nociceptin, also referred to as orphanin FQ, is believed to be the endogenous ligand for the ORL1. Nociceptin, when injected intracerebroventricularly to mice, produced hyperalgesia in behavioral tests. Recent studies have demonstrated the presence of ORL1 transcript in the spinal cord, and ORL1-like immunoreactivity has been localized to nerve fibers and somata throughout the spinal cord. Here, we report the localization of nociceptin-like immunoreactivity to fiber-like elements of the superficial layers of the rat dorsal horn by immunohistochemical techniques. Whole-cell recordings from substantia gelatinosa neurons in transverse lumbar spinal cord slices of 22-26-day-old rats showed that exogenous nociceptin at low concentrations (100-300 nM) depressed excitatory postsynaptic potentials evoked by stimulation of dorsal rootlets without causing an appreciable change of resting membrane potentials and glutamate- evoked depolarizations. At a concentration of 1 μM, nociceptin hyperpolarized substantia gelatinosa neurons and suppressed spike discharges. The hyperpolarizing and synaptic depressant action of nociceptin was not reversed by the known opioid receptor antagonist naloxone (1 μM). Our result provides evidence that nociceptin-like peptide is concentrated in nerve fibers of the rat dorsal horn and that it may serve as an inhibitory transmitter within the substantia gelatinosa.

The large conductance calcium dependent potassium (BKCa) channels are expressed in a large variety of cell types including neurons where they modulate excitability and action potential shape. Within the enteric nervous system, stretch-sensitive BKCa channels are expressed on intrinsic primary afferent neurons (IPANs) where they decrease the neurons’ excitability during intestinal contractions. A major determinant of peristalsis is slow excitatory neurotransmission (sEPSPs) within the IPAN to IPAN sensory network, and we wondered whether such transmission might also alter BKCa channel opening. All experiments were performed on longitudinal-muscle myenteric preparations prepared from jejunal segment taken from freshly euthanized adult male Swiss Webster mice. With the myenteric plexus exposed by microdissection, BKCa channel activity was recorded in cell-attached mode via the patch clamp technique. BKCa channel activity was recorded before and after presynaptic electrical stimulation, which was designed to evoke postsynaptic sEPSPs. The morphotype was verified by intracellular injection of a marker dye (neurobiotin). In addition, a blocker and opener were used to identify the effects of BKCa currents on IPAN properties. Analysis of unitary channel recordings revealed increased BKCa open probability (NPo) at fixed trans-patch potentials following sEPSPs. All BKCa channels were independently voltage sensitive with increased NPo during patch depolarisation. Analysis of whole-cell experiments also revealed BKCa channels have a significant effect on the undershoot amplitude of action potentials, and the rate at which IPANs repolarise. This study demonstrates that sEPSPs within the enteric nervous system modulate the function of BKCa channels in IPANs adding to the mechanistic understanding of enteric synaptic transmission and providing a potential target for therapeutic modulation of enteric nervous system excitability. / Thesis / Master of Science (MSc)

Sensory feedback possesses the possibility of adding a new dimension to many applications, including, but not limited to, prosthetics and surgical robots for improved control, virtual reality for incorporation of another sense, and phantom limb pain reduction for amputees. Electrotactile stimulation provides a compact, light-weight, energy efficient, highly responsive, and non-invasive option for sensory feedback; however, it has been found to commonly elicit unnatural or uncomfortable sensations for the user. To address this issue, this thesis was designed to test the impact of the different electrotactile stimulus parameters – current amplitude and polarity, pulse width, frequency, and waveform – on the user’s perceived sensation and afferent neural response. The relationship between sensation and neural response was also analysed. The aim of this thesis was to create guidelines to assist in the design and use of electrotactile stimulation. Neural data and matching psychophysical data from one healthy subject and purely psychophysical data from three others were gathered while applying electrotactile stimulations of different parameter combinations on the dorsal side of the hand or lower arm. Significant (p < 0.05) correlations and differences were found in all three relationships between electrotactile stimulus parameters, primary afferent response, and perceived sensation. Current (specifically negative) or pulse width control in monophasic waveforms were deemed most appropriate in applications that relay information through varying intensity. However, monophasic waveforms produced more discomfort, rendering biphasic waveforms more suitable when mild, local, and more natural sensations are of greater importance. Surprisingly, the data also suggested higher sensitivity to positive currents. While lower values of current amplitude and pulse width increased neural spike count, stimulus frequency could reliably control neural firing at all tested frequencies. Spike counts were moderately to strongly correlated with perceived intensity; however, practically identical neural responses could elicit different sensations. High current pulses at low frequencies induced spikes with the shortest latency – but with greater risk of discomfort. Due to limitations in sample size, generalisability is limited, and this thesis should be considered a pilot study to guide future investigations. The results suggest that recording from single and multiple afferent nerve fibres simultaneously would improve the understanding of the neural population response to electrotactile stimuli. Moreover, the one-to-one neural response to electrotactile stimuli raised the question of whether an electrotactile stimulation based on a natural spike pattern could replicate the original sensation. A future study testing this hypothesis may find a new approach to designing painless electrotactile stimulations for sensory feedback use. / Sensorisk återkoppling har möjlighet att drastiskt förbättra många användningsområden, t.ex. genom att bidra till enklare kontroll av proteser och kirurgiska robotar, mer verklighetstrogna VR-spel, och minskade fantomsmärtor hos patienter med amputeringar. Elektrisk hudstimulering erbjuder ett kompakt, lätt, energisnålt, hög-responsivt och icke-invasivt alternativ för sensorisk återkoppling – dock framkallar denna metod ofta onaturliga och obehagliga förnimmelser för användaren. Detta examensarbete bemöter detta problem genom att undersöka effekten av stimuleringsparametrar som strömstyrka, pulsbredd, frekvens och vågform på försökspersonens upplevda förnimmelse samt den afferenta nervresponsen. Även relationen mellan förnimmelse och nervrespons analyserades. Examensarbetets ändamål var att skapa riktlinjer för att förenkla designen och användandet av elektrisk hudstimulering. Nervdata med matchande psykofysiska data samlades från en frisk försöksperson, samt enbart psykofysiska data från tre andra, under olika elektriska hudstimuleringar (med varierande parametervärden) på handens dorsala sida eller på underarmen. Signifikanta (p < 0.05) korrelationer och skillnader fanns i alla tre relationer mellan parametrarna för elektrisk hudstimulering, primärafferent respons och upplevd förnimmelse. Kontroll av ström (i synnerhet negativ) eller pulsbredd i monofasisk vågform visade sig vara mest fördelaktigt i applikationer där information kommuniceras till användaren genom att variera den upplevda intensiteten. Dock skapade denna vågform mer obehag, och bifasiska vågformer bedömdes mer passande då milda, lokala, och mer naturliga förnimmelser är av högre värde. Positiv ström upplevdes, förvånande, starkare än negativ. Vid låga värden på ström och pulsbredd var dessa faktorer viktiga gällande antalet aktionspotentialer (AP), men stimuleringsfrekvensen kunde kontrollera antalet AP vid alla frekvenser. Mängden AP var måttligt till starkt korrelaterad med upplevd intensitet – samtidigt kunde praktiskt taget identiska nervresponser vara kopplade till olika förnimmelser. Lågfrekvent stimulering med hög ström hade kortast latenstid, men högre risk för obehag. P.g.a. lågt antal försökspersoner är generaliserbarheten begränsad och detta examensarbete bör beaktas som en förstudie för att guida framtida forskning. Resultaten från denna studie antyder att en tydligare bild av populationsresponsen skulle kunna skapas genom samtidig läsning av ett flertal enskilda nervfibrer samtidigt. Det faktum att varje stimulering kunde ge upphov till en AP väckte frågan: kan ett stimuleringsmönster baserat på en naturlig nervrespons återskapa den ursprungliga förnimmelsen? En studie som testar denna hypotes har möjligheten att finna ett nytt tillvägagångssätt för att skapa smärtfri elektrisk hudstimulering för sensorisk återkoppling.

Les travaux entrepris au cours de cette thèse ont eu pour but d'étudier la modulation de la transmission synaptique inhibitrice au niveau des réseaux moteurs spinaux, à la fois au cours du développement et après lésion de la moelle épinière. Le nouveau-né présente des activités motrices spontanées qui jouent un rôle important dans la maturation des muscles et des réseaux de neurones de la moelle épinière. Dans une première étude, nous avons identifié l'un des mécanismes impliqués dans la genèse de ces activités chez le rat nouveau-né in vitro. Nous avons démontré que l'activation des récepteurs GABAᴀ au niveau des terminales d'afférences primaires joue un rôle majeur dans le déclenchement et la propagation de ces activités spontanées. Dans une deuxième étude, nous avons testé la robustesse des dépolarisations de nature GABAergique enregistrées in vitro, c'est-à-dire leur dépendance vis-à-vis des paramètres du milieu de perfusion. Nous avons démontré que l'action dépolarisante des neurotransmetteurs GABA/glycine au niveau des motoneurones et celle du GABA au niveau des terminales d'afférences primaires ne sont pas dues à une fourniture énergétique insuffisante. La dernière étude a été consacrée à la modulation de la transmission synaptique inhibitrice après lésion de la moelle épinière. Nous avons montré que l'activation des récepteurs 5-HT2 (R5-HT2), particulièrement celle de l'isoforme 5-HT2ᴀ, renforce le poids synaptique inhibiteur via une hyperpolarisation du potentiel d'équilibre des ions chlorure (ECl) et une augmentation d'expression de KCC2 au niveau de la membrane des motoneurones. / The aim of this thesis was to explore the modulation of the inhibitory synaptic transmission within the spinal motor networks, both during development and after SCI. Spontaneous movements are an ubiquitous feature of fetal and infant behavior. They provide signals that are important for the development of muscles and the assembly of neuronal networks in the spinal cord. In a first study, we characterized one of the mechanisms underlying spontaneous motor behaviors in the in vitro spinal cord preparation isolated from neonatal rats. We demonstrated that the GABA is playing a key role in promoting spontaneous activity through primary afferent depolarizations which reach firing threshold. In the second part of my thesis, we tested the robustness of the in vitro GABAergic depolarizations and their dependence on the aCSF parameters. We demonstrated that during development the depolarizing actions of GABA/glycine on motoneurons and GABA on primary afferent terminals are not due to inadequate energy supply. In the last part of my thesis, we focused on the modulation of the inhibitory synaptic transmission following SCI. We demonstrated that activation of the 5-HT2 receptors, particularly the 5-HT2ᴀ subtype, strengthens inhibitory synaptic transmission to spinal motoneurons by hyperpolarizing the reversal potential of Cl- ions (ECl) and by increasing the cell-membrane expression of KCC2. This phenomenon reduces spasticity after SCI in rats. Upregulation of KCC2 function by targeting 5-HT2ᴀ receptors therefore opens new therapeutic strategies for the treatment of spasticity following SCI.