A crucial question in drug addiction research concerns whether the varying reports of dopamine-independent and dopamine-dependent motivation can be integrated. According to one theory, the prior drug history of a subject — that is to say, whether they have received minimal or chronic drug exposure — determines whether opiate motivation is dependent upon the brainstem tegmental pedunculopontine nucleus (TPP) or dopamine neurotransmission. The biological analogue of this change is thought to be a switch in the signalling properties (from hyperpolarizing to depolarizing) of ventral tegmental area (VTA) gamma-aminobutyric acid subtype-A (GABAA) receptors. In this thesis, I demonstrate that the mechanisms underlying opiate motivation can be selected artificially by manipulating the signalling properties of VTA GABAA receptors, irrespective of the past drug history of the subject. Furthermore, I suggest that these same VTA GABAA receptors also play a similar role in controlling ethanol motivation. Indeed, the mechanisms underlying ethanol motivation can be doubly dissociated in a manner similar to that observed with opiates. However, whereas opiate motivation is TPP-dependent in the drug-naive state, I found that ethanol motivation was dependent on dopamine neurotransmission (via the D2 receptor) in drug-naive animals. Conversely, ethanol motivation was TPP-dependent in ethanol-deprived mice (as opposed to opiate motivation being dopamine-dependent in opiate-deprived animals). These effects are consistent with a VTA GABAA receptor switching mechanism identical to the one seen in the case of opiate motivation.

A crucial question in drug addiction research concerns whether the varying reports of dopamine-independent and dopamine-dependent motivation can be integrated. According to one theory, the prior drug history of a subject — that is to say, whether they have received minimal or chronic drug exposure — determines whether opiate motivation is dependent upon the brainstem tegmental pedunculopontine nucleus (TPP) or dopamine neurotransmission. The biological analogue of this change is thought to be a switch in the signalling properties (from hyperpolarizing to depolarizing) of ventral tegmental area (VTA) gamma-aminobutyric acid subtype-A (GABAA) receptors. In this thesis, I demonstrate that the mechanisms underlying opiate motivation can be selected artificially by manipulating the signalling properties of VTA GABAA receptors, irrespective of the past drug history of the subject. Furthermore, I suggest that these same VTA GABAA receptors also play a similar role in controlling ethanol motivation. Indeed, the mechanisms underlying ethanol motivation can be doubly dissociated in a manner similar to that observed with opiates. However, whereas opiate motivation is TPP-dependent in the drug-naive state, I found that ethanol motivation was dependent on dopamine neurotransmission (via the D2 receptor) in drug-naive animals. Conversely, ethanol motivation was TPP-dependent in ethanol-deprived mice (as opposed to opiate motivation being dopamine-dependent in opiate-deprived animals). These effects are consistent with a VTA GABAA receptor switching mechanism identical to the one seen in the case of opiate motivation.

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Mu opioid receptors (MOPs) in the pain pathway contribute to morphine analgesia. Morphine also stimulates reward/reinforcement through disinhibition of dopaminergic (DA) neurones in the ventral tegmental area (VTA), an effect implicated in its abuse and dependence. We hope to develop approaches to achieve sustained analgesia without affecting reward by exploiting differential MOP signalling mechanisms in the pain and reward pathways. MOPs, delta opioid receptors (DOPs) and β-arrestin2 (BAR2) are all necessary components of the signalling complex in nociceptive neurones for morphine analgesic tolerance; c-Src (a tyrosine kinase), thought to couple to MOP receptors through BAR2 has also been implicated. To investigate opioid receptor signalling in response to morphine we used a variety of different techniques that included behavioural measures of nociception, reinforcement and locomotion and electrophysiological methods to study DRG neurones from the pain pathway and brain slices containing VTA neurones. This study in mice confirms that morphine administered subcutaneously (SC) causes analgesia, analgesic tolerance, and has psychomotor effects leading to enhanced locomotion and reinforcement. In VTA neurones morphine and the selective MOP receptor agonist DAMGO caused concentration-dependent inhibition of the frequency of IPSCs. All these actions of morphine were absent from MOP-/- mice. Morphine exhibited reduced potency as 1) an analgesic, 2) stimulator of locomotion, 3) a reinforcer in CPP and 4) an inhibitor of sIPSC frequency, when applied to MOP+/- mice or their VTA neurones. Morphine analgesic tolerance developed faster and to a greater extent in MOP+/- mice than in WT mice. DOP-/- mice exhibited morphine analgesia with less tolerance, as did BAR2-/- mice. BAR2-/- mice also exhibited reduced morphine locomotion and an increased sensitivity to morphine reinforcement. Morphine tolerance was absent from BAR2-/-//DOP-/- mice. The inhibition of sIPSC frequency by morphine was reduced in BAR2+/- and BAR2-/- VTA neurones. Dasatinib and PP2 (c-Src tyrosine kinase inhibitors) prevented the development of morphine tolerance in WT and MOP+/- mice and dasatinib caused its reversal in the latter. The drugs had no significant analgesic effect alone. Dasatinib did not affect morphine preference or locomotor activation. PP2 reduced morphine’s inhibition of sIPSC frequency. As c-Src inhibition does not appear to alter the psychomotor effects produced by morphine and it acts to reduce morphine analgesic tolerance. We believe that cSrc is an attractive target to prevent the development of morphine analgesic tolerance without affecting hedonic homeostasis.

Despite the advances made in our understanding of the molecular mechanistic actions of general anesthetics very little is known about the in vivo neural circuits involved in creating the state of general anesthesia. To date the common consensus is that general anesthetics act ubiquitously within the CNS. Recently, (Devor and Zalkind, 2001) have reported that microinjections of pentobarbital (PB) into a discrete brainstem focal area of conscious rats induced a classical, reversible general anesthesia-like behavioral state. The authors concluded that this area, termed the mesopontine tegmental anesthesia area (MPTA), may be important for the induction of general anesthesia. The purpose of the present project was to study the neurophysiological basis of the analgesia, which accompanied the state of general anesthesia induced by PB microinjections into the MPTA that was reported by (Devor and Zalkind, 2001). Here, sensory inflow via the spinothalamic tract (STT), a classical spinal nociceptive pathway in the rat, was assessed using single neuron extracellular recording techniques before, during and after microinjections of PB into the MPTA. Spontaneous firing rate (SFR), antidromic firing index (FI) and sciatic as well as sural nerve-evoked responses (Sc-, Su-ER) of STT neurons in isoflurane-anesthetized rats were quantified before as well as 2, 15, 30 and 60 min following bilateral microinjections of either PB (200 micrograms/side) or vehicle control solution (Vh, 1 microliter/side) into the MPTA. The group mean SFR, FI as well as magnitudes of Sc-, Su-ER of STT neurons were significantly and reversibly reduced following PB microinjections compared to corresponding baseline measurements. There were no significant changes in any of the three parameters following microinjections of Vh compared to the pre-microinjection baseline responses. The results from this study indicate that analgesia, which occurs during the anesthesia-like state following microinjections of PB into the MPTA, may be due to attenuation of sensory inflow through the STT. The suppression of STT neurons likely occurs via direct and/or indirect descending pathways from the MPTA to the spinal cord. This study provides the first direct electrophysiological evidence for the analgesia caused by PB microinjections into the rat MPTA.

The mechanisms underlying the generation of rapid eye movement (REM) sleep are poorly understood. Despite a lack of direct support, neurons maximally active during REM sleep (REM sleep-active) located in the pedunculopontine tegmental nucleus (PPTn) are hypothesized to generate this state and its component phenomenology. This hypothesis has never been directly tested, since the results of selectively inhibiting this cell-group have never been determined. Using microdialysis, electrophysiology, histochemical and pharmacological methods in freely-behaving rats (n=22) instrumented for sleep-wake state and respiratory muscle recordings, I selectively inhibited REM sleep-active PPTn neurons. Contrary to the prevailing hypothesis, I showed that REM sleep-active PPTn neurons suppress REM sleep by limiting the frequency of its onset. These neurons also shape the impact of REM sleep on breathing. REM sleep-active PPTn neurons restrain behavioural activation of upper-airway musculature during REM sleep, while depressing breathing rate and respiratory activation of the upper-airway musculature across sleep-wake-states.

The mechanisms underlying the generation of rapid eye movement (REM) sleep are poorly understood. Despite a lack of direct support, neurons maximally active during REM sleep (REM sleep-active) located in the pedunculopontine tegmental nucleus (PPTn) are hypothesized to generate this state and its component phenomenology. This hypothesis has never been directly tested, since the results of selectively inhibiting this cell-group have never been determined. Using microdialysis, electrophysiology, histochemical and pharmacological methods in freely-behaving rats (n=22) instrumented for sleep-wake state and respiratory muscle recordings, I selectively inhibited REM sleep-active PPTn neurons. Contrary to the prevailing hypothesis, I showed that REM sleep-active PPTn neurons suppress REM sleep by limiting the frequency of its onset. These neurons also shape the impact of REM sleep on breathing. REM sleep-active PPTn neurons restrain behavioural activation of upper-airway musculature during REM sleep, while depressing breathing rate and respiratory activation of the upper-airway musculature across sleep-wake-states.

El núcleo pedunculopóntico tegmental (PPTg) es un grupo de neuronas (predominantemente colinérgicas) situado en el tronco del encéfalo, que se ha implicado en diversas funciones cognitivas. No obstante, el papel que juega el PPTg en los procesos cognitivos no está del todo claro; por ejemplo, no se conoce si su influencia en estos aspectos depende del nivel de dificultad de la tarea, del número de opciones de respuesta disponibles de la misma o de su dependencia de los sistemas fronto-estriatales; por otra parte, no se sabe si su integridad es necesaria para la consolidación de la memoria. Para contestar a estas cuestiones, se ha realizado un trabajo compuesto por tres experimentos en el que se analizan los efectos de la lesión del PPTg sobre: 1) la adquisición del condicionamiento de evitación activa de dos sentidos (EV2), una tarea dependiente del estriado dorsal, en dos condiciones diferentes de dificultad, 2) la retención a largo plazo, al cabo de 10 días, de este mismo tipo de condicionamiento; y 3) el aprendizaje de dos tareas espaciales dependientes de los sistemas fronto-estriatales ventrales, que implican únicamente dos opciones de respuesta, y que difieren en cuanto a su grado de dificultad (la tarea de no-alternancia forzada o DMTP y la tarea de alternancia forzada o DNMTP, ambas en un laberinto en T). En el experimento I, los animales eran sometidos a lesión electrolítica bilateral del PPTg y posteriormente recibían dos sesiones de EV2 separadas por 10 días; la mitad de los sujetos eran entrenados con un estímulo condicionado (EC) de 10 segundos de duración (condición de baja dificultad) y la otra mitad con un EC de 3 segundos (condición de alta dificultad). En el experimento II, los sujetos recibían una sesión de entrenamiento en la tarea de EV2 de mayor dificultad e, inmediatamente después, eran sometidos a lesión electrolítica del PPTg; 10 días más tarde se evaluaba el nivel de retención de la misma. En el experimento III, se les realizaba a los animales una lesión química (con ácido iboténico) bilateral en el PPTg y posteriormente eran entrenados en una tarea de DMTP demorada 15 segundos y a otra de DNMTP demorada 15 segundos. Si alcanzaban el criterio de aprendizaje de esta segunda tarea, eran también entrenados en una tarea de DNMTP demorada 30 segundos. Los principales resultados de estos trabajos ponen de manifiesto que: 1) la integridad del PPTg es necesaria para la adquisición de la tarea de EV2, tanto en condiciones de alta como de baja demanda; 2) la participación de este núcleo no es necesaria para la retención a largo plazo de la EV2, ni siquiera en los primeros estadios del proceso de consolidación; 3) el PPTg participa en la adquisición de tareas espaciales dependientes de sistemas fronto-estriatales, incluso cuando implican dos únicas opciones de respuesta; 4) las variaciones en el nivel de demanda de la tarea influyen en el efecto deteriorante de la lesión del PPTg sobre la adquisición de la EV2 y de las tareas dependientes de sistemas fronto-estriatales, pero no son un determinante crítico para el mismo; y 5) los déficits observados tras la lesión del PPTg en la tarea de EV2 y en las dos pruebas espaciales (el DMTP y el DNMTP) no parecen ser atribuibles a cambios en la motricidad espontánea, en el nivel de motivación, en la reactividad emocional, ni en la sensibilidad al dolor; sino a un deterioro cognitivo que podía estar mediado por la alteración del funcionamiento córtico-estriatal. / The pedunculopontine tegmental nucleus (PPTg) is a neuronal group (predominantly cholinergic) located in the brain stem, which has been involved in several cognitive functions. However, the role of the nucleus in these cognitive functions is not well understood. For instance, it is not known whether its influence on cognition depends on the level of difficulty of the task, on the number of response options available, or on its dependence of fronto-striatal systems. On the other hand, it is not known if its integrity is necessary for memory consolidation. In order to answer these questions, we have carried out a work composed of three experiments analyzing the effects of PPTg lesions on: 1) the acquisition of two-way active avoidance conditioning, a task that depends on dorsal striatal systems, under two conditions differing in task difficulty; 2) the long-term retention (10 days) of the same conditioning task; and 3) the acquisition of two spatial tasks that depend on fronto-ventral striatal systems, that involve only two response options, and that differ in their difficulty level (delayed matching-to-position task or DMTP, and delayed non-matching-to-position task, or DNMTP). In Experiment I, the animals were subjected to bilateral electrolytic lesions of the PPTg and were thereafter trained in a two-way active avoidance task (2 sessions separated by 10 days). Half of the subjects were trained using a conditioned estimulus of 10 second duration (low difficulty condition), while the other half were trained with a conditioned stimulus lasting 3 seconds (high difficulty condition). In Experiment II, the animals received a training sessions in two-way active avoidance under the high difficulty condition, and, immediately after this session were subjected to an electrolytic lesion of the PPTg. Long-term retention was tested 10 days later. In Experiment III, the animals were subjected to bilateral chemical lesions (ibotenic acid) of the PPTg, and were trained in a DMTP task with a 15-second delay, and in a DNMTP task with a 15-second delay. The animals that reached the learning criterion for the latter were trained in a second DNMTP using a delay of 30 seconds. The main results obtained in the present work indicate that: 1) the integrity of the PPTg integrity is necessary for the acquisition of two-way active avoidance in all the conditions tested; 2) this nucleus is not necessary for the long-term retention of two-way active avoidance, even in the first stages of the consolidation process; 3) the PPTg participates in the acquisition of spatial tasks that depend on fronto-ventral striatal systems even when they only involve two response options; 4) variations in task demand can affect the disruptive effects of PPTg lesions on the acquisition of two-way active avoidance and of spatial tasks that depend on fronto-ventral striatal systems, but they are not a critical factor in determining this disruption; and 5) the learning deficits observed after PPTg lesions do not seem to be attributable to changes in spontaneous locomotion, in motivational level, in emotional reactivity or in pain sensitivity; rather they seem to be induced by cognitive deterioration that could be mediated by the alteration of functioning of the cortico-striatal systems.

Despite the advances made in our understanding of the molecular mechanistic actions of general anesthetics very little is known about the in vivo neural circuits involved in creating the state of general anesthesia. To date the common consensus is that general anesthetics act ubiquitously within the CNS. Recently, (Devor and Zalkind, 2001) have reported that microinjections of pentobarbital (PB) into a discrete brainstem focal area of conscious rats induced a classical, reversible general anesthesia-like behavioral state. The authors concluded that this area, termed the mesopontine tegmental anesthesia area (MPTA), may be important for the induction of general anesthesia. The purpose of the present project was to study the neurophysiological basis of the analgesia, which accompanied the state of general anesthesia induced by PB microinjections into the MPTA that was reported by (Devor and Zalkind, 2001). Here, sensory inflow via the spinothalamic tract (STT), a classical spinal nociceptive pathway in the rat, was assessed using single neuron extracellular recording techniques before, during and after microinjections of PB into the MPTA. Spontaneous firing rate (SFR), antidromic firing index (FI) and sciatic as well as sural nerve-evoked responses (Sc-, Su-ER) of STT neurons in isoflurane-anesthetized rats were quantified before as well as 2, 15, 30 and 60 min following bilateral microinjections of either PB (200 micrograms/side) or vehicle control solution (Vh, 1 microliter/side) into the MPTA. The group mean SFR, FI as well as magnitudes of Sc-, Su-ER of STT neurons were significantly and reversibly reduced following PB microinjections compared to corresponding baseline measurements. There were no significant changes in any of the three parameters following microinjections of Vh compared to the pre-microinjection baseline responses. The results from this study indicate that analgesia, which occurs during the anesthesia-like state following microinjections of PB into the MPTA, may be due to attenuation of sensory inflow through the STT. The suppression of STT neurons likely occurs via direct and/or indirect descending pathways from the MPTA to the spinal cord. This study provides the first direct electrophysiological evidence for the analgesia caused by PB microinjections into the rat MPTA.

Despite the advances made in our understanding of the molecular mechanistic actions of general anesthetics very little is known about the in vivo neural circuits involved in creating the state of general anesthesia. To date the common consensus is that general anesthetics act ubiquitously within the CNS. Recently, (Devor and Zalkind, 2001) have reported that microinjections of pentobarbital (PB) into a discrete brainstem focal area of conscious rats induced a classical, reversible general anesthesia-like behavioral state. The authors concluded that this area, termed the mesopontine tegmental anesthesia area (MPTA), may be important for the induction of general anesthesia. The purpose of the present project was to study the neurophysiological basis of the analgesia, which accompanied the state of general anesthesia induced by PB microinjections into the MPTA that was reported by (Devor and Zalkind, 2001). Here, sensory inflow via the spinothalamic tract (STT), a classical spinal nociceptive pathway in the rat, was assessed using single neuron extracellular recording techniques before, during and after microinjections of PB into the MPTA. Spontaneous firing rate (SFR), antidromic firing index (FI) and sciatic as well as sural nerve-evoked responses (Sc-, Su-ER) of STT neurons in isoflurane-anesthetized rats were quantified before as well as 2, 15, 30 and 60 min following bilateral microinjections of either PB (200 micrograms/side) or vehicle control solution (Vh, 1 microliter/side) into the MPTA. The group mean SFR, FI as well as magnitudes of Sc-, Su-ER of STT neurons were significantly and reversibly reduced following PB microinjections compared to corresponding baseline measurements. There were no significant changes in any of the three parameters following microinjections of Vh compared to the pre-microinjection baseline responses. The results from this study indicate that analgesia, which occurs during the anesthesia-like state following microinjections of PB into the MPTA, may be due to attenuation of sensory inflow through the STT. The suppression of STT neurons likely occurs via direct and/or indirect descending pathways from the MPTA to the spinal cord. This study provides the first direct electrophysiological evidence for the analgesia caused by PB microinjections into the rat MPTA. / Pharmaceutical Sciences, Faculty of / Graduate