The neurotransmitter dopamine (DA) represents a neural substrate for positive motivation as its spatiotemporal distribution across the brain is responsible for goaldirected behavior and learning reward associations. The critical determinant of DA release throughout the brain is the firing pattern of DA-producing neurons. Synchronized bursts of spikes can be triggered by sensory stimuli in these neurons, evoking phasic release of DA in target brain areas to drive reward-based reinforcement learning and behavior. These bursts are generated by NMDA-type glutamate receptors (NMDARs). This dissertation reports a novel form of long-term potentiation (LTP) of NMDARmediated excitatory transmission at DA neurons as a putative cellular substrate for changes in DA neuron firing during reward learning. Patch-clamp electrophysiological recording from DA neurons in acute brain slices from young adult rats demonstrated that synaptic NMDARs exhibit LTP in an associative manner, requiring coordinated pre- and postsynaptic burst firing. Ca2+ signals produced by postsynaptic burst firing needed to be amplified by preceding metabotropic neurotransmitter inputs to effectively drive plasticity. Activation of NMDARs themselves was also necessary. These two coincidence detectors governed the timingdependence of NMDAR plasticity in a manner analogous to the timing rule for cuereward learning paradigms in behaving animals. Further mechanistic study revealed that PKA, but not PKC, activity gated LTP induction by regulating the magnitude of Ca2+ signal amplification via the inositol 1,4,5-triphospate (IP3) receptor and release of Ca2+ from intracellular stores. Plasticity of NMDARs was input specific and appeared to be expressed postsynaptically, but was not associated with a change in NMDAR subunit stoichiometry. LTP of NDMARs was DA-independent, and was specific for NMDARs: the same induction protocol produced long-term depression of AMPA receptors. NMDARs that had undergone LTP could be depotentiated in a spike-conditional manner, consistent with active unlearning. Finally, repeated, in vivo amphetamine experience dramatically increased facilitation of spike-evoked Ca2+ signals, which in turn drove enhanced plasticity. NMDAR plasticity thus represents a potential neural substrate for conditioned DA neuron burst responses to environmental stimuli acquired during reward-based learning as well a novel therapeutic target for intervention-based therapy of addictive disorders. / text

A computational model of a midbrain dopamine neuron was extended in this study to include a response to random excitatory afferent input by incorporating the receptor components AMPA and NMDA. In a diagonal band where average glutamatergic and tonic gabaergic input is roughly balanced, both single spike firing and bursting can be observed. Simulated SK channel block strengthens the correlation between pattern and rate and increases the number of spikes fired in bursts by increasing the spikes per burst. A simulated doubling of the AMPA/NMDA ratio leads to a frequency increase that becomes more prominent at high firing rates, and an increase in the percent spikes fired in bursts. Changes in pattern and rate are poorly correlated in the model. Manipulations of the neuron greatly depend on the background level of synaptic inputs, suggesting that interpretation of population data from dopamine neurons requires taking variability into account rather than averages.

GABA is the major inhibitory neurotransmitter in the central nervous system. It binds to two types of receptors –ionotropic GABAA and metabotropic GABAB. The GABAA receptor directly gates a Clionophore that causes hyperpolarization in mature excitatory neurons while GABAB receptor mediates a slower hyperpolarizing response via G-protein coupled receptor (GPCR) activated potassium channels. This signaling mechanism gets further complicated by the heterogeneous GABA receptor subunit composition that influences the response kinetics in the postsynaptic membrane. In this thesis, the focus has been to decipher the role of GABAA receptors in relation to cellular excitability in the subiculum under physiological and pathophysiological conditions. The subiculum, considered as the output structure of hippocampus, modulates information flow from hippocampus to various cortical and sub-cortical areas and has been implicated in learning and memory, rhythm generation and various neurological disorders. It gates hippocampal activity with its well orchestrated and fine tuned intrinsic and local network properties. Over the years many studies have shown the involvement of subiculum in temporal lobe epilepsy where it forms the focal point of epileptiform activities with altered cellular and network properties. The subiculum is characterized by the presence of a significant population of burst firing neurons that lead local epileptiform activity. By virtue of its bursting nature and recurrent connections, it is a potential site for seizure generation and maintenance. Epileptiform activities are dynamic in nature and change temporally and spatially according to the alterations in electrophysiological properties of neurons. Transitions to different electrical activities in neurons following a prolonged challenge with epileptogenic stimulus have been shown in other brain structures, but not in the subiculum. Considering the importance of the subicular burst firing neurons in the propagation of epileptiform activity to the entorhinal cortex, we have explored the phenomenon of electrophysiological phase transitions in the burst firing neurons of the subiculum in an in vitro brain slice model of epileptogenesis. Whole-cell patch clamp and extracellular field recordings revealed a distinct phenomenon in the subiculum wherein an early hyperexcitable phase was followed by a late suppressed phase upon continuous perfusion with epileptogenic 4-amino pyridine and magnesium-free medium. The late suppressed phase was characterized by inhibitory post-synaptic potentials (IPSPs) in pyramidal excitatory neurons and bursting activity in local fast spiking interneurons at a frequency of 0.1-0.8 Hz. The IPSPs were mediated by GABAA receptors that coincided with excitatory synaptic inputs to attenuate action potential discharge. These IPSPs ceased following a cut between the CA1 and subiculum. Our results suggest the importance of feedforward inhibition in the suppression of epileptiform activity in subiculum to mediate a homeostatic response towards the induced hyper-excitability. GABA release from presynaptic nerve endings activates postsynaptic GABAA receptors, which evoke faster phasic inhibitory postsynaptic currents (IPSCs) and non-inactivating inhibitory tonic current, mediated through extrasynaptic GABAA receptors. These receptors are heteropentameric GABA-gated channels assembled from 19 possible subunits (α1-6, β1-3, γ1-3, δ, π, ρ1-3, θ, and ε). The 2 major subunits involved in tonic GABAA currents in the hippocampus are α5 and δ subunits. Tonic GABAA receptor mediated inhibitory current plays an important role in neuronal physiology as well as pathophysiology such as mood disorders, insomnia, epilepsy, autism spectrum disorders and schizophrenia. While the alterations of various electrical properties due to tonic inhibition have been studied in neurons from different regions, its influence on intrinsic subthreshold resonance in pyramidal excitatory neurons having hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is not known. In the present study, we show the involvement of α5βγ GABAA receptors in mediating picrotoxin sensitive tonic current in subicular pyramidal neurons using known pharmacological agents that target specific GABAA receptor subunits. We further investigated the contribution of tonic conductance in regulating subthreshold electrophysiological properties using current clamp and dynamic clamp experiments. Our experiments suggest that tonic GABAergic inhibition can actively modulate subthreshold properties of subicular pyramidal neurons including resonance due to HCNchannels that may potentially alter the response dynamics in an oscillating neuronal network.

Les mouvements masticatoires sont générés par un réseau neuronal localisé dans le tronc cérébral connu sous le nom de générateur de patron central (GPC). De plus en plus d’évidences dans la littérature associent le noyau sensoriel principal du trijumeau (NVsnpr) au cœur rythmogène du GPC masticatoire, bien qu’il soit traditionnellement considéré comme un relais sensoriel au thalamus. La présente étude amène une nouvelle preuve de connectivité fonctionnelle entre le NVsnpr et le noyau moteur du trijumeau (NVmt) contenant les motoneurones (MNs) innervant les différents muscles masticatoires. Nos résultats indiquent que les neurones projetant vers NVmt sont situés dans le ¾ dorsal du NVsnpr. La stimulation électrique dans le NVsnpr dorsal évoque des réponses synaptiques excitatrices multiphasiques dans les MNs trigéminaux tandis que l'application locale de BAPTA, connue pour induire une activité rythmique dans les neurones du NVsnpr, évoqua aussi une activité rythmique dans les MNs, supportant davantage la relation fonctionnelle entre ces deux noyaux en termes de transmission de rythme. En imagerie calcique, la stimulation électrique de NVsnpr évoquait des réponses calciques dans les MNs situées principalement dans la région dorsolatérale contenant les MNs des muscles de fermeture et révéla un patron spécifique de connectivité entre les deux noyaux. L'organisation des projections semblait dépendre de manière critique de la localisation dorso-ventrale du site de stimulation au sein du NVsnpr. La principale tendance observée concernait la région DL de NVmt recevant des inputs du NVsnpr dorsal (R1 et R2), alors que la région ventromédiane de NVmt recevait plutôt des inputs de R2 et R3, qui représentent la majeure partie de la région intermédiaire du NVsnpr. Cette étude confirme et développe les expériences antérieures en explorant la nature physiologique et la topographie fonctionnelle de la connectivité entre NVsnpr et NVmt. / Masticatory movements are generated by a brainstem neuronal network known as the central pattern generator (CPG). Increasing evidence associate the trigeminal main sensory nucleus (NVsnpr) to the rhythmogenic heart of the masticatory CPG, despite the fact that it is conventionally seen as a sensory relay to the thalamus. The present study provides new evidence of a functional connectivity between NVsnpr and the trigeminal motor nucleus (NVmt), known to contain all the motoneurons (MNs) innervating jaw muscles. Our results indicate that neurons projecting to NVmt are located in the dorsal ¾ region of NVsnpr. Electrical stimulation of the dorsal NVsnpr induced multiphasic excitatory synaptic responses in trigeminal MNs while BAPTA application, which causes NVsnpr neurons to fire rhythmically, also induced rhythmic firing in some MNs, further emphasizing the functional relationship between these two nuclei in terms of rhythm transmission. In our calcium imaging experiments, electrical stimulation of NVsnpr evoked calcium responses in MNs located mainly in the jaw-closing region of NVmt and revealed a specific pattern of connectivity between the two nuclei. The organization of the projections seemed to depend critically on the dorsoventral location of the stimulation site within NVsnpr. The dorsolateral region of NVmt received mainly inputs from the dorsal NVsnpr (R1 and R2), whereas the ventromedial region of NVmt was found to receive inputs from R2 and R3 which account for the major part of the intermediate division of the NVsnpr. This study confirms and develops earlier experiments by exploring the physiological nature and functional topography of the connectivity between NVsnpr and NVmt that was demonstrated in the past with neuroanatomical techniques.

Ionenkanäle leiten Ionenströme über neuronale Membranen, wodurch Aktionspotentiale erzeugt und weitergeleitet werden. Sie spielen eine zentrale Rolle bei der Regulierung der Erregbarkeit und des Aktivierungsverhaltens von Neuronen. KCNQs sind eine wichtige Familie von spannungsgesteuerten Kaliumkanälen; ihre Dysfunktion kann zu verschiedenen neurologischen Krankheiten führen, einschließlich Erkrankung an Epilepsie und Taubheit. Es wurde gezeigt, dass KCNQ2 und KCNQ3 den M-Strom verantwortlich sind. Letzterer ist für die Regulierung des repetitiven Feuerns von Pyramidenzellen entscheidend. Im Gegensatz zu KCNQ2, ist die funktionelle Bedeutung von KCNQ3 noch nicht aufgeklärt. In dieser Arbeit zeigen wir mittels extrazellulärer Elektrophysiologie in vivo, dass bei konstitutiven Kcnq3 Knockoutmäusen die hippokampalen Pyramidenzellen vermehrt burstartig feuern. Außerdem weisen diese Tiere eine verminderte Spike-Frequenz-Anpassung auf und die Wahrscheinlichkeit des Burst-Feuerns während zwei verschiedener Oszillationen – Theta gegen Nicht-Theta – kann nicht mehr unterscheiden werden. Des Weiteren zeigen Kcnq3-Knockout- Pyramidenzellen während der Theta-Oszillation weder eine dominante Phasenpräferenz, noch eine Koordination ihrer Burst-Feuerung. Die Thetawellen Phasenpräzision tritt weiterhin bei dem vorübergehend verstärkten Feuern auf. Das räumliche selektive Feuern von mutmaßlichen Ortszellen blieb auch bei den Knockout-Mäusen erhalten, aber es ist hauptsächlich vom Burst- Feuern abhängig. Diese Studie zeigt, dass der KCNQ3-Ionenkanal eine wichtige Rolle bei der Regulierung der neuronalen Erregbarkeit und der Informationsverarbeitung spielt, und gibt damit Einblicke in die Bedeutsamkeit der KCNQ3-Ionenkanäle bezüglich der neurologischen Störungen. / Ion channels conduct ion flows across neuronal membrane whereby action potential is generated and propagated. They play a central role in regulating the excitability and firing behavior of a neuron. Among them, the KCNQs present a prominent family of voltage-gated potassium channels. Dysfunction of KCNQ2–5 channels can lead to varied neurological diseases including early onset epilepsy and deafness. In cortex and hippocampus, KCNQ2 and KCNQ3 have been demonstrated to underlie the non-inactivating M-current critical for controlling the repetitive firing of pyramidal cells. However, the functional significance of KCNQ3, unlike that of KCNQ2, remains elusive. Here, by applying in vivo extracellular electrophysiology in Kcnq3 constitutive knockout mice and the wild-type littermates, we find that hippocampal pyramidal cells lacking KCNQ3 exhibit increased burst firing. Moreover, the spike frequency adaptation of their bursts is diminished, and the burst propensity during two different field oscillations – theta versus non-theta – becomes indistinguishable. During theta oscillations, Kcnq3 knockout pyramidal cells no longer display unimodal phase preference and do not coordinate their burst firing. But phase advancement along successive theta cycles continues to occur at times of transiently intensified firing. The selective firing of place cells is largely preserved in the knockout while mainly relying on bursts. These results suggest that KCNQ3 channels indeed play a significant and specific role in regulating the neurons’ excitability and information processing, thus providing crucial mechanistic insights into the relevance of the KCNQ3 channels in neurological disorders.

No description available.

Dopamine (DA) neurons of the substantia nigra pars compacta (SNc) have a key role in regulation of voluntary movement control. Their death is a hallmark of Parkinson’s disease, characterised by inhibited motor control, including muscle rigidity and tremor. Excitatory input to SNc-DA neurons is primarily from the subthalamic nucleus, and in PD these afferents display a higher frequency firing, as well as increased burst firing, which could cause increased excitatory activity in SNc-DA neurons. NMDA receptors (NMDARs) bind the excitatory neurotransmitter glutamate, and are essential for learning and memory. In SNc-DA neurons, NMDARs have a putative triheteromeric subunit arrangement of GluN1 plus GluN2B and/or GluN2D. Wild type (WT) mice, and those lacking the gene for GluN2D (Grin2D-null), were used to explore its role in various aspects of DA neuronal function and dysfunction using patch-clamp electrophysiology, viability assaying, and immunofluorescence. Pharmacological intervention using subunit-specific inhibitors ifenprodil and DQP-1105 on elicited NMDAR-EPSCs suggested a developmental shift from primarily GluN2B to GluN2B/D. Activity dependent regulation was assessed by high frequency burst stimulation of glutamatergic afferents: in comparison to controls, significant downregulation of NMDARs was observed in SNc-DA neurons, though no differences were observed based on genotype. This regulatory function may be a neuroprotective or homeostatic response. Ambient extracellular glutamate elicits tonic NMDAR activity in SNc-DA neurons, which may be important for maintaining basal levels of excitability: the role of GluN2D was assessed by recording the deflection in baseline current caused by application of competitive NMDAR antagonist D-AP5. There was a significantly larger NMDAR-mediated current in WT vs Grin2D-null mice, indicating that GluN2D has a role in binding ambient glutamate. Dysfunction of glutamate uptake could be a secondary pathophysiological occurrence in the SNc, leading to increased ambient glutamate: the effect of this was explored by application of the competitive glutamate transporter blocker TBOA. Here, the NMDAR-mediated portion of this current was significantly higher in WT mice in comparison to Grin2D-null. Interestingly, dose-response data obtained from bath application of NMDA showed significantly larger currents in Grin2D-null animals vs WT, but only at the top of the response curve (~1-10 mM), which may indicate a capability for larger conductance in Grin2D-null animals at high NMDAR saturation due to replacement of GluN2D with GluN2B. GluN2D may therefore be neuroprotective, by attenuating peak current flow in response to very high agonist concentrations. Lastly, GluN2D has been found to decrease NMDAR open probability under hypoxic conditions, potentially conferring resistance to hypoxia / ischemia related excitotoxicity. Therefore, low (15% O2 / 80% N2 / 5% CO2) vs high (95% O2 / 5% CO2) oxygen conditions were used along with immunofluorescent propidium iodide cell death assaying and immunofluorescent labeling for DA neurons in order to compare levels of DA neuronal death in the SNc based on oxygen status and genotype. Whilst there was a significant submaximal effect based on O2 status, genotype did not confer a practical resistance under these conditions. In summary, NMDARs have diverse roles in SNc-DA neurons which may both serve to maintain normal function and protect the cell against potentially pathological conditions.

The subiculum is a structure that forms a bridge between the hippocampus and the entorhinal cortex (EC) in the brain, and plays a major role in the memory consolidation process. It consists of different types of pyramidal neurons. Based on their firing behavior, these excitatory neurons are classified into strong burst firing (SBF), weak burst firing (WBF) and regular firing (RF) neurons. In the first part of the work, morphological differences in the different neuronal subtypes was explored by biocytin staining after classifying the neurons based on the differences in electrophysiological properties. Detailed morphological properties of these three neuronal subtypes were analyzed using Neurolucida neuron reconstruction method. Unlike the differences in their electrophysiological properties, no difference was found in the morphometric properties of these neuronal subtypes. In the second part of the thesis, experimental results on spike- timing- dependent plasticity (STDP) at the proximal excitatory inputs on the subicular pyramidal neurons of the juvenile (P15-P19) rat are described. The STDP was studied in the WBF and RF neurons. Causal pairing of a single EPSP with a single back propagating action potential (bAP) at a time interval of 10 ms failed to induce plasticity. However, increasing the number of bAPs in such EPSP-bAP pair to three at 50 Hz (bAP burst) induced LTD in both, the RF, as well as the WBF neurons. Increasing the frequency of action potentials to 150 Hz in the bAP burst during causal pairing also induced LTD in both the neuronal subtypes. However, all other STDP related experiments were performed only with the bAP bursts consisting of 3 bAPs evoked at 50 Hz. Amplitude of the causal pairing induced LTD decreased with increasing time interval between EPSP and the bAP burst. Reversing the order of the EPSP and the bAP burst in the pair induced LTP only with a short time interval of 10 ms. This finding is in contrast to most of the reports on excitatory synapses, wherein the pre-before post (causal) pairing induced LTP and vice-versa. The results of causal and anti-causal pairing were used to plot the STDP curve for the WBF neurons. In the STDP curve observed in these synapses, LTD was observed upto a causal time interval of 30 ms, while LTP was limited to 10 ms time interval. Hence, the STDP curve was biased towards LTD. These results reaffirm the earlier observations that the relative timing of the pre- and postsynaptic activities can lead to multiple types of STDP curves. Next, the mechanism of non-Hebbian LTD was studied in both, the RF and WBF neurons. The involvement of calcium in the postsynaptic neuron in plasticity induction was studied by chelating intracellular calcium with BAPTA. The results indicate that the LTD induction in WBF neurons required postsynaptic calcium, while LTD induction in the RF neurons was independent of postsynaptic calcium. Paired pulse ratio (PPR) experiments suggested the involvement of a presynaptic mechanism in the induction of LTD in the RF neurons, and not in the WBF neurons since the PPR was unaffected by the induction protocol only in the WBF neurons. LTD induction in the WBF neurons required activity of the NMDA receptors since LTD was not observed in the presence of the NMDA receptor blocker in the WBF neurons, while it was unaffected in the RF neurons. However, the RF neurons required the activity of L-type calcium channels for plasticity induction, since LTD was affected in the presence of the L-type calcium channel blockers, although the WBF neurons did not require the L-type calcium channel activity for plasticity induction. Hence, in addition to a non-Hebbian STDP curve, a novel mechanism of LTD induction has been reported, where L-type calcium channels are involved in a synaptic plasticity that is expressed via change in the release probability. The findings on the STDP in subicular pyramidal neurons may have strong implications in the memory consolidation process owing to the central role of the subiculum and LTD in it.