The Effects of NMDA Subunit Composition on Calcium Influx and Spike Timing-Dependent Plasticity in Striatal Medium Spiny Neurons

Calcium through NMDA receptors (NMDARs) is necessary for the long-term potentiation (LTP) of synaptic strength; however, NMDARs differ in several properties that can influence the amount of calcium influx into the spine. These properties, such as sensitivity to magnesium block and conductance decay kinetics, change the receptor's response to spike timing dependent plasticity (STDP) protocols, and thereby shape synaptic integration and information processing. This study investigates the role of GluN2 subunit differences on spine calcium concentration during several STDP protocols in a model of a striatal medium spiny projection neuron (MSPN). The multi-compartment, multi-channel model exhibits firing frequency, spike width, and latency to first spike similar to current clamp data from mouse dorsal striatum MSPN. We find that NMDAR-mediated calcium is dependent on GluN2 subunit type, action potential timing, duration of somatic depolarization, and number of action potentials. Furthermore, the model demonstrates that in MSPNs, GluN2A and GluN2B control which STDP intervals allow for substantial calcium elevation in spines. The model predicts that blocking GluN2B subunits would modulate the range of intervals that cause long term potentiation. We confirmed this prediction experimentally, demonstrating that blocking GluN2B in the striatum, narrows the range of STDP intervals that cause long term potentiation. This ability of the GluN2 subunit to modulate the shape of the STDP curve could underlie the role that GluN2 subunits play in learning and development

Intrinsic and synaptic adaptations in neuronal ensembles following recall of appetitive associative memories: investigations in the striatum and prefrontal cortex with the Fos-GFP mouse

Learned associations between rewarding stimuli and environmental cues which predict their availability play an important role in guiding behaviour. These learned associations are thought to be encoded by neuroadaptations in disperse sets of strongly activated neurons, termed neuronal ensembles, located throughout motivationally-relevant brain areas. However to date, the nature of the adaptations which occur selectively on neuronal ensembles encoding appetitive associative memories remain largely unknown. Using the Fos-GFP mouse, which expresses green fluorescent protein (GFP) in recently activated neurons, we investigated the intrinsic and synaptic excitability of neurons activated following exposure to stimuli associated with food (sucrose) or drug (cocaine) exposure. We observed that in the nucleus accumbens (NAc) shell, but not orbitofrontal cortex, neurons activated following exposure to a food-associated stimulus were more intrinsically excitable than surrounding, non-activated neurons. These neurons also demonstrated increased spontaneous excitatory transmission suggestive of potentiated synaptic strength. Following extinction of the food-cue association, NAc shell neurons activated following cue exposure were no longer more excitable than surrounding neurons. This suggests that the intrinsic excitability of striatal neurons activated by a food-associated cue is dynamically modulated by changes in associative strength. We also examined the intrinsic excitability of striatal neurons (including neurons in the NAc shell, core and dorsal striatum) activated by cocaine-associated stimuli. Interestingly, NAc shell neurons activated by cocaine-associated stimuli were not more excitable compared to the surrounding neurons regardless of extinction learning experience, possibly indicating differences between drug and food conditioning. Similar results were obtained for dorsal striatal neurons. However, NAc core neurons activated by cocaine-associated stimuli displayed an enhanced excitability which persisted following extinction, indicating that core and shell neuronal ensembles differentially encode the cocaine associative memories. Overall, by selectively recording from stimuli-activated neurons, this work reveals novel adaptations at the intrinsic and synaptic levels on neuronal ensembles following appetitive learning with both food and drug rewards

The effects of reward devaluation on cue-evoked modulation of sucrose seeking and neuronal ensemble plasticity in nucleus accumbens

Animals must learn the relationship between food and the environmental cues that predict their availability for the successful procurement of nutrient sources. These cues can gain powerful control over food seeking, but these cue-evoked behaviours must remain flexible and updated upon changes in internal states such as the perceived desirability of food. Recalling these cue-food associations activate subsets of neurons termed ‘neuronal ensembles’ in motivationally relevant brain areas such as the striatum. However, how neuronal ensembles are recruited and physiologically modified following the update of these learned associations has not fully elucidated. To investigate this, we examined the effects of reward devaluation on ensemble plasticity at the levels of recruitment, excitability, and synaptic physiology in sucrose conditioned Fos-GFP mice that express green fluorescent protein (GFP) in recently activated neurons. Neuronal ensemble activation patterns and their physiology were examined using immunohistochemistry and ex vivo electrophysiology, respectively. First, devaluation via four days of ad libitum sucrose consumption, but not caloric satiation, attenuated the ability of the cue to evoke sucrose seeking. Thus, changes in the hedonic, incentive value of sucrose, and not caloric need drove cue-induced sucrose seeking. Also, devaluation attenuated the cue’s ability to recruit a neuronal ensemble in nucleus accumbens (NAc), but not dorsal striatum. Next, devaluation prevented the cue from recruiting a hyper-excitable, GFP+ ensemble in the NAc, but did not alter the physiology of excitatory synapses on these GFP+ neurons. Our findings provide new insights into how updates in the hedonic value of sucrose critically modulates the flexibility of sucrose seeking and recruitment of ensembles with an altered excitability phenotype in the NAc shell

The impact of psychostimulant administration during development on adult brain functions controlling motivation, impulsivity and cognition.

ADHD pharmacotherapy uses methylphenidate (MPH), D-amphetamine (D- amph), two psychostimulants targeting dopamine transporters, or atomoxetine (ATX), specifically targeting norepinephrine transporters. We have assessed the pharmacological mechanisms of these three drugs on the in vitro efflux of neurotransmitters in rat prefrontal cortex (PFC) and striatal slices as well as on the in vivo electrical activities of PFC pyramidal neurons, striatal medium spiny neurons, ventral tegmental area dopamine neurons or dorsal raphe nucleus serotonin neurons, using single cell extracellular electrophysiological recording techniques. We have also tested whether chronic methylphenidate treatment, during either adolescence or adulthood, could have long-lasting consequences on body growth, depression and neuronal functions. Release experiments showed that all ADHD drugs induce dose-dependent dopamine efflux in both the PFC and striatum, with different efficacies, while only D- amph induced cortical norepinephrine efflux. Atomoxetine induced an unexpected massive dopamine outflow in striatal regions, by mechanisms that depend on physiological parameters. Our electrophysiological studies indicate that all three drugs equally stimulate the excitability of PFC pyramidal neurons, in basal and NMDA-evoked conditions, when administered acutely (3 mg/kg). While the electrophysiological effects elicited by psychostimulants may be dependent on D1 receptor activation, those induced by atomoxetine relied on different mechanisms. In the ventral tegmental area (VTA), methylphenidate (2 mg/kg), but not atomoxetine, induced firing and burst activity reductions, through dopamine D2 autoreceptor activation. Reversal of such effects (eticlopride 0.2 mg/kg) revealed an excitatory effect of methylphenidate on midbrain dopamine neurons that appear to be dependent on glutamate pathways and the combination of D1 and alpha-1 receptors. Finally, acute intraperitoneal psychostimulant injections increased vertical locomotor activity as well as NMDA2B protein expression in the striatum. Some animals chronically treated with intraperitoneal administrations (methylphenidate 4 mg/kg/day or saline 1.2 ml/kg/day) showed decreased body weight gain. Voluntary oral methylphenidate intake induces desensitisation to subsequent intravenous methylphenidate challenges, without altering dopamine D2 receptor plasticity. Significant decreases in striatal NMDA2B protein expression were observed in animals chronically treated. After adolescent MPH treatment, midbrain dopaminergic neurons do not display either desensitisation or sensitisation to intravenous methylphenidate re-challenges. However, partial dopamine D2 receptor desensitisation was observed in midbrain dopamine neurons. Using behavioural experiments, cross-sensitisation between adolescent methylphenidate exposure and later-life D-amphetamine challenge was observed. Significant decreases in striatal NMDA2B protein expression were observed in animals chronically treated, while striatal medium spiny neurons showed decreased sensitivities to locally applied NMDA and dopamine. While caffeine is devoid of action on baseline spike generation and burst activity of dopamine neurons, nicotine induces either firing rate enhancement, firing rate reduction, or has no consequences. Adolescent methylphenidate treatment leads to decreased neuronal sensitivities to the combination of nicotine, MPH and eticlopride, compared to controls. Finally, nicotine partially prevented D-amphetamine-induced increase of rearing activities. Our results show that increases in the excitability of PFC neurons in basal conditions and via NMDA receptor activation may be involved in the therapeutic response to ADHD drugs. Long-term consequences were observed after psychostimulant exposure. Such novel findings strengthen the mixed hypothesis in ADHD, whereby both dopamine and glutamate neurotransmissions are dysregulated. Therefore, ADHD therapy may now focus on adequate balancing between glutamate and dopamine

Modulation of NMDA Receptor Currents in Rat Substantia Nigra

Dopamine receptor signalling is essential for normal basal ganglia function but in Parkinson’s Disease (PD) substantia nigra (SNc) dopaminergic (DAergic) neurons degenerate with consequent loss of dopamine signalling. SNc DAergic neurons express D2 autoreceptors (D2Rs) that have been shown to mediate inhibition of NMDA responses in both hippocampus and striatum while Gascoupled adenosine A2A receptors (A2ARs) have the potential to counteract the action of Gai -coupled D2Rs. Here I tested whether D2R activation with ropinirole, a D2 receptor agonist currently used in PD therapy, modulates DAergic neuron NMDA responses in the SNc along with other proteins in the cell. Whole-cell patch clamp recordings were made from DAergic neurons in the SNc of acute midbrain slices from young (P7, P21 and P28) rats. DAergic neurons were identified by the presence of a prominent hyperpolarisation-activated inward current (in P7 rats, amplitude, 178 ± 5 pA; activation time constant, 797 ± 77 ms; mean ± SEM, N = 19) in response to a voltage step from -60 to -120 mV. In P7 and P28 rats, upon application of 200 nM ropinirole, the steady state NMDA current was not significantly changed suggesting D2-R activation may not modulate NMDARs in neonatal rat SNc. In addition, an A2AR agonist, CGS21680, and an A2AR antagonist, SCH 58621 were applied in the presence and absence of ropinirole to test for any A2AR – D2R interaction. Upon A2A-R activation, the NMDA-R current increased (P = 0.002, N = 16). Furthermore, to establish the effects of PKA on NMDA-R responses, 2.5µM Forskolin was introduced. It produced a statistically significant increase in NMDA-R current (NMDA: 419 ± 78pA; NMDA+ Forskolin: 515 ± 54pA, N=13). To determine whether the lack of effect of the D2-R agonist on NMDA-R response might be due to a low resting concentration of cAMP in the cell, forskolin was introduced to increase the levels of cAMP prior to introducing ropinirole. However, following addition of D2-R agonist after forskolin treatment, the NMDA-R current changed by only 11% (N=12). Intracellular tyrosine kinases, Src and Fyn have shown modulatory potential on NMDA-Receptors (NMDA-R) that is governed by the balance between kinase and phosphatase activity. Inhibiting Src kinase activity with PP2 and Src-I1 decreased the NMDA-R inward current however no such effect was seen in the presence of the interfering peptides suggesting a lack of direct interaction between Src/Fyn kinase and NMDA-Rs. Furthermore, ERK1/2 inhibitor, Ulixertinib, decreased the NMDA-R current suggesting an involvement in receptor modulation. Similar results were obtained in the presence of a CaMKII inhibitor CN21

Mechanisms of Basal Ganglia Development

Animals must respond to external cues and changes in internal state by modifying their behavior. The basal ganglia are a collection of subcortical nuclei that contribute to action selection by integrating sensorimotor, limbic and reward information to control motor output. In early life, however, animals display distinct behavioral responses to risk and reward and enhanced vulnerability to neuropsychiatric disease. This arises from the postnatal maturation of brain structures such as the striatum, the main input nucleus of the basal ganglia. Here, using biochemical, electrophysiological and behavioral approaches in transgenic mice, I have explored the molecular and circuit mechanisms that control striatal maturation. In Chapter 1, I begin by reviewing the structure, physiology and function of the basal ganglia, with an emphasis on the striatum. I then describe the existing literature on the development and maturation of striatal neurons and their afferents. In Chapter 2, I review the molecular mechanisms of macroautophagy, a lysosomal degradation pathway that has recently been implicated in the regulation of neurotransmission, including its contribution to neuronal development, neurotransmitter release, and postsynaptic function. The subsequent chapters can be split into two themes. In the first, encompassing chapters 3 and 4, I characterize the postnatal maturation of striatal physiology and define circuit mechanisms that control this process. In Chapter 3, I demonstrate that dopamine (DA) neurotransmission in the striatum initiates the maturation of striatal projection neuron (SPN) intrinsic excitability. I show that DA signaling leads to the maturation of SPN excitability via increased activity of the potassium channel, Kir2. Interestingly, introduction of DA beginning in adulthood could not rescue SPN hyperexcitability while it could during the juvenile period. In Chapter 4, I characterize the maturation of cholinergic interneurons (ChIs) in the striatum and describe the biophysical mechanisms that drive increases in spontaneous activity that occur in ChIs during postnatal development. Finally, I show that the functional maturation of ChIs leads to changes in DA release during the postnatal period. The second theme includes Chapters 5 and 6, in which I explore the role of macroautophagy in striatal function and development. In chapter 5, I used biochemical approaches to show that autophagic flux is suppressed postnatally in the striatum due to increased signaling through the kinase activity of the mammalian target of rapamycin. In Chapter 6, I generated conditional knockouts of Atg7, a required macroautophagy gene, in different populations of SPNs and find that macroautophagy plays cell-type specific roles in SPN physiology. In one subtype of SPNs, macroautophagy regulates intrinsic excitability via degradation of Kir2 channels, which is the first demonstration of macroautophagic control of neuronal excitability. Finally, in Chapter 7, I conclude with a general discussion, where I highlight themes in the molecular and circuit mechanisms of striatal maturation and their implication for neurodevelopmental disease

Contributions to models of single neuron computation in striatum and cortex

A deeper understanding is required of how a single neuron utilizes its nonlinear subcellular devices to generate complex neuronal dynamics. Two compartmental models of cortex and striatum are accurately formulated and firmly grounded in the experimental reality of electrophysiology to address the questions: how striatal projection neurons implement location-dependent dendritic integration to carry out association-based computation and how cortical pyramidal neurons strategically exploit the type and location of synaptic contacts to enrich its computational capacities.Neuronale Zellen transformieren kontinuierliche Signale in diskrete Zeitserien von Aktionspotentialen und kodieren damit Perzeptionen und interne Zustände. Kompartiment-Modelle werden formuliert von Nervenzellen im Kortex und Striatum, die elektrophysiologisch fundiert sind, um spezifische Fragen zu adressieren: i) Inwiefern implementieren Projektionen vom Striatum ortsabhängige dendritische Integration, um Assoziationens-basierte Berechnungen zu realisieren? ii) Inwiefern nutzen kortikale Zellen den Typ und den Ort, um die durch sie realisierten Berechnungen zu optimieren

Post-translational SUMOylation dynamically regulates voltage-gated potassium channel, Kv4.2

The family of voltage-gated Kv4 ion channels (Kv4.1-3) mediates the transient A-type potassium currents, IA, and is an important regulator of neuronal signaling. Aberrations in Kv4 channel expression and/or function are associated with several disease states, including chronic pain, epilepsy, Alzheimer’s disease, Huntington’s disease and major depressive disorder. Kv4 channels exist as ternary complexes with potassium channel interacting proteins and dipeptidyl peptidase-like proteins. Multiple ancillary proteins also associate with the Kv4 ternary complex throughout its lifetime. Little is known about the regulation of protein-protein interactions within Kv4 macromolecular complexes. Small ubiquitin-like modifier (SUMO) is a peptide that is post-translationally conjugated to lysine (K) residues on target proteins. This post-translational modification dynamically regulates protein-protein interactions. It can either promote or prevent a given interaction. This dissertation research investigated if/how post-translational SUMOylation moderated Kv4.2 protein-protein interactions to tune IA. Kv4.2 has several putative SUMOylation sites. Two conserved sites were examined in this work: K437 and K579. SUMOylating K579 increased IA when Kv4.2 existed in the ternary complex but decreased IA when Kv4.2 was expressed alone. Studies to identify the mechanism indicated that K579 SUMOylation increased IA by promoting ternary complex recycling after endocytosis, most likely by blocking an interaction with a ubiquitin ligase and thereby reducing a ubiquitin lysosome sorting signal. In contrast, when Kv4 was not incorporated into a ternary complex, K579 SUMOylation blocked an unknown protein-protein interaction that altered channel gating to reduce IA. SUMOylation at the second site, K437, had no effect when Kv4.2 was incorporated into the ternary complex, but increased the insertion of electrically silent channels when Kv4.2 was expressed alone. The mechanism underpinning increased surface expression was not examined. These dissertation findings were the first to demonstrate that Kv4.2 can be SUMOylated to regulate IA, that SUMOylation modulates Kv4.2 internalization and that the effect of SUMOylation depends upon the available interactome