Synthesis and discovery of the putative cognitive enhancer BRS-015: effect on glutamatergic transmission and synaptic plasticity

This thesis is concerned with the discovery of a novel heterocyclic compound – BRS-015, its synthesis and an analysis of its effects on excitatory synaptic transmission at a major pathway in the brain. BRS-015 is related to the natural product clausenamide, which has been shown to facilitate synaptic transmission. As such, clausenamide and related analogues may possess therapeutic potential as memory enhancing drugs, which are in urgent need of development due to the increasing numbers of patients diagnosed with memory disorders and for which there is no current effective therapy. BRS-015 was synthesized using a novel approach to the core structure of clausenamide involving an intramolecular acylal cyclisation reaction, which has not previously been reported. The first section of the thesis opens with a description of the discovery, structure and biological activity of clausenamide and discussion of previous synthetic strategies adopted by a number of research groups and attempts to classify these into the varying approaches towards the central core of clausenamide. The second section describes the structure of the rat brain and the types of processes involved in memory formation, as well as the neurophysiological assays used to investigate synaptic transmission and plasticity. The second group of chapters describes our own approach to the core of clausenamide and the synthesis of BRS-015, with a detailed discussion of the structural analysis and investigation of the intramolecular acylal cyclisation reaction used during the synthetic process. The third chapter describes the neurophysiological assays used in our investigations into the effects of BRS-015, which was tested against glutamatergic synaptic transmission and plasticity in acute rat hippocampal slices. BRS-015 was shown to reversibly enhance the amplitude of AMPA receptor mediated EPSCs recorded from CA3 pyramidal neurones and evoked by dentate stimulation. When tested in the presence of selective glutamate receptor antagonists, BRS-015 did not have this powerful enhancing effect on kainate or NMDA receptor mediated EPSCs. In addition, BRS-015 increased the amplitude of glutamate-evoked currents in CA3 pyramidal neurones and did not alter short-term synaptic plasticity but facilitated the induction of mossy fibre LTP, with little effect at associational/commissural synapses. BRS-015 has striking enhancing properties on AMPA receptor mediated synaptic transmission at mossy fibre synapses either by directly interacting with AMPA receptors or via indirect modulation, the mechanisms of which could lead to synapse strengthening

Morphological Correlates of Long-term Potentiation and Ageing in the Hippocampus of Rats

This thesis has examined age-dependent changes in neural plasticity in rat hippocampal dentate gyrus using unbiased morphological techniques at light and electron microscope level. It considers whether there is a morphological basis to explain why some aged animals sustain, whilst others fail to sustain, potentiation in the dentate gyrus of the hippocampus after unilateral induction of LTP, 45 minutes after stimulation of the perforant pathway. Previous data in young adult rats (5 months old) have demonstrated that the morphology of dendritic spines and synapses within the hippocampus is altered at 10-30 minutes, and 24 hrs following LTP induction. The data obtained in the present study suggest that differences in spine and synaptic morphological parameters appear to be correlated with the ability to maintain LTP in the aged rats. Those maintaining LTP had a tendency to longer spine length, and a decrease in spine and synaptic densities, there was a significant increase in the number of complex axospinous perforated synapses. Here, in young rats, LTP resulted in a significant increase in the density of spine synapses and total synaptic density. The mean spine density was also higher in the stimulated hemisphere, but spine length decreased. However, there was a significant increase in the number of bifurcating spines and axospinous perforated complex synapses in the stimulated compared to the contralateral control hemisphere. An age-dependent comparison indicated that spine density and synaptic densities are significantly higher in the younger rats, but spine length was significantly greater in the aged rats. LTP does not seem to cause morphological changes per se at the time examined post potentiation. However, an important finding of the present study is that the percentage of axospinous complex perforated synapses is significantly higher in the stimulated hemisphere of aged and young rats that sustained LTP compared to those that did not. The percentage of branched spines and simple axospinous perforated synapses is significantly higher in both the stimulated & unstimulated hemisphere of aged rats that failed to sustain LTP. Therefore a proportion of the branched spines and perforated synapses would appear to be the result of high frequency stimulation, rather than LTP induction per se

Mechanisms of amyloid-beta cytotoxicity in hippocampal network function : rescue strategies in Alzheimer's disease

The origin and nature of cognitive processes are strongly associated with synchronous rhythmic activity in the brain. Gamma oscillations that span the frequency range of 30–80 Hz are particularly important for sensory perception, attention, learning, and memory. These oscillations occur intrinsically in brain regions, such as the hippocampus, that are directly linked to memory and disease. It has been reported that gamma and other rhythms are impaired in brain disorders such as Alzheimer’s disease, Parkinson’s disease, and schizophrenia; however, little is known about how these oscillations are affected. In the studies contained in this thesis, we investigated a possible involvement of toxic Amyloid-beta (Aβ) peptide associated with Alzheimer’s disease in degradation of gamma oscillations and the underlying cellular mechanismsin rodent hippocampi. We also aimed to prevent possible Aβ- induced effects by using specially designed molecular tools known to reduce toxicity associated with Aβ by interfering with its folding and aggregation steps. Using electrophysiological techniques to study thelocal field potentials and cellular properties in the CA3 region of the hippocampus, we found that Aβ in physiological concentrations acutely degrades pharmacologically- induced hippocampal gamma oscillations in vitro in a concentration- and time- dependent manner. The severity of degradation also increased with the amount of fibrillar Aβ present. We report that the underlying cause of degradation of gamma oscillations is Aβ-induced desynchronization of action potentials in pyramidal neurons and a shift in the equilibrium of excitatory-inhibitory synaptic transmission. Using specially designed molecular tools such as Aβ-binding ligands and molecular chaperones, we provide evidence that Aβ-induced effects on gamma oscillations, cellular firing, and synaptic dynamics can be prevented. We also show unpublished data on Aβ effects on parvalbumin-positive baskets cells or fast-spiking interneurons, in which Aβ causes an increase in firing rate during gamma oscillations. This is similar to what is observed in neighboring pyramidal neurons, suggesting a general mechanism behind the effect of Aβ. The studies in this thesis provide a correlative link between Aβ-induced effects on excitatory and inhibitory neurons in the hippocampus and extracellular gamma oscillations, and identify the Aβ aggregation state responsible for its toxicity. We demonstrate that strategies aimed at preventing peptide aggregation are able to prevent the toxic effects of Aβ on neurons and gamma oscillations. The studies have the potential to contribute to the design of future therapeutic interventions that are aimed at preserving neuronal oscillations in the brain to achieve cognitive benefits for patients

Role of galanin in synaptic transmission and plasticity in the CA1 area of the rodent hippocampus

Galanin is believed to be co-released with acetylcholine by neurones projecting from the medial septum and nucleus ofMeynert to the hippocampus in rodents. Galanin inhibits acetylcholine and glutamate release, thereby depressing excess neuronal excitability in the brain. Although this effect established galanin as an endogenous neuroprotective substance, released only during high frequency neuronal firing, it may also explain why it impairs memory and cognition in vivo. The sustained increase in glutamatergic synaptic strength following high frequency stimulation of hippocampal neurones, a phenomenon termed long-term potentiation (LTP), has been widely recognised as a model ofthe synaptic changes that may underlie learning and memory in vertebrates. It may thus be predicted that the physiological action ofgalanin at the cellular level would be to depress LTP, thereby causing an impairment in mnemonic processes mediated by the hippocampus. Experiments were designed to address aspects ofthis hypothesis, namely: (1) in vitro characterisation of the effect ofgalanin agonists and antagonists on synaptic transmission and plasticity in the CA1 area of rodent hippocampus and (2) investigation of glutamatergic synaptic plasticity in galanin knockout mice and their wild-type littermates. Exogenous galanin induced a dose-dependent increase in the slope of baseline fEPSPs, which appeared to be dependent on the pathways from CA3 to CA1 being intact, but it did not have any effect on paired-pulse facilitation ratios (PPF) in low concentration. However, in higher concentration, galanin induced a significant decrease in PPF in intact slices. In CA3-hemisected hippocampal slices the aforementioned effects did not occur. The effect of galanin on LTP and long-term depression (LTD) of glutamate mediated synaptic transmission in apical and basal dendrites of CA1 pyramidal neurones were investigated using both intracellular and extracellular recording techniques in vitro. LTP induced in either apical or basal dendrites of CA1 pyramidal neurones by different paradigms was significantly inhibited by galanin. Galanin also inhibited LTP in hippocampal slices prepared from wild-type mice. This effect was reversible by the known galanin antagonist, galantide (Ml5). Galanin did not affect isolated pure NMDA receptor-mediated postsynaptic potentials or the loss of spike frequency adaptation and increase in input resistance evoked by metabotropic glutamate receptor activation, indicating that its inhibition of LTP was downstream ofthese receptors. Galanin applied had no effect the expression of LTP indicating that galanin may inhibit LTP by interfering with kinase activity necessary for the induction of LTP, e.g. protein kinase C. Galanin did not affect the induction of LTD. Subsequent studies in the galanin-null transgenic mice yielded no effect on synaptic strength or paired pulse facilitation ratios. Galanin gene deletion caused a significant impairment of LTP, which was only observed in basal dendrites, the magnitude ofwhich increased with age. The underlying molecular mechanism for this impairment might be a significantly faster saturation of synaptic plasticity in the mutant mice in vivo, compared to wild-type mice. No effects of galanin were noted in mutant mice. This could suggest a developmental loss of galanin-responsive cells concomitant with global galanin gene deletion. In summary, galanin seems to have a modulatory effect on excitatory neurotransmitters in the hippocampus, such as glutamate, thereby delaying the neurodegenerative effect of age. The research described in this thesis is deemed of importance in biomedical research of drug therapy for protection against neurodegenerative disease

Slow Inhibition and Inhibitory Recruitment in the Hippocampal Dentate Gyrus

L’hippocampe joue un rôle central dans la navigation spatiale, la mémoire et l’organisation spatio-temporelle des souvenirs. Ces fonctions sont maintenues par la capacité du gyrus denté (GD) de séparation des patrons d'activité neuronales. Le GD est situé à l’entrée de la formation hippocampique où il reconnaît la présence de nouveaux motifs parmi la densité de signaux afférant arrivant par la voie entorhinale (voie perforante). Le codage parcimonieux est la marque distinctive du GD. Ce type de codage est le résultat de la faible excitabilité intrinsèque des cellules granulaires (CGs) en combinaison avec une inhibition locale prédominante. En particulier, l’inhibition de type « feedforward » ou circuit inhibiteur antérograde, est engagée par la voie perforante en même temps que les CGs. Ainsi les interneurones du circuit antérograde fournissent des signaux GABAergique aux CGs de manière presque simultanée qu’elles reçoivent les signaux glutamatergiques. Cette thèse est centrée sur l’étude des interactions entre ces signaux excitateurs de la voie entorhinale et les signaux inhibiteurs provenant des interneurones résidant dans le GD et ceci dans le contexte du codage parcimonieux et le patron de décharge en rafale caractéristique des cellules granulaires. Nous avons adressé les relations entre les projections entorhinales et le réseau inhibitoire antérograde du GD en faisant des enregistrements électrophysiologiques des CG pendant que la voie perforante est stimulée de manière électrique ou optogénétique. Nous avons découvert un nouvel mécanisme d’inhibition qui apparait à délais dans les CGs suite à une stimulation dans les fréquences gamma. Ce mécanisme induit une hyperpolarisation de longue durée (HLD) et d’une amplitude prononce. Cette longue hyperpolarisation est particulièrement prolongée et dépasse la durée d’autres types d’inhibition transitoire lente décrits chez les CGs. L’induction de HLD crée une fenêtre temporaire de faible excitabilité suite à laquelle le patron de décharge des CGs et l’intégration d’autres signaux excitateurs sont altérés de manière transitoire. Nous avons donc conclu que l’activité inhibitrice antérograde joue un rôle central dans les processus de codage dans le GD. Cependant, alors qu’il existe une multitude d’études décrivant les interneurones qui font partie de ce circuit inhibiteur, la question de comment ces cellules sont recrutées par la voie entorhinale reste quelque peu explorée. Pour apprendre plus à ce sujet, nous avons enregistré des interneurones résidant iii dans la couche moléculaire du GD tout en stimulant la voie perforante de manière optogénétique. Cette méthode de stimulation nous a permis d’induire la libération de glutamate endogène des terminales entorhinales et ainsi d’observer le recrutement purement synaptique d’interneurones. De manière surprenante, les résultats de cette expérience démontrent un faible taux d’activation des interneurones, accompagné d’un tout aussi faible nombre total de potentiels d’action émis en réponse à la stimulation même à haute fréquence. Ce constat semble contre-intuitif étant donné qu’en générale on assume qu’une forte activité inhibitrice est requise pour le maintien du codage parcimonieux. Tout de même, l’analyse des patrons de décharge des interneurones qui ont été activés a fait ressortir la prééminence de trois grands types: décharge précoce, retardée ou régulière par rapport le début des pulses lumineux. Les résultats obtenus durant cette thèse mettent la lumière sur l’important conséquences fonctionnelles des interactions synaptique et polysynaptique de nature transitoire dans les réseaux neuronaux. Nous aimerions aussi souligner l’effet prononcé de l’inhibition à court terme du type prolongée sur l’excitabilité des neurones et leurs capacités d’émettre des potentiels d’action. De plus que cet effet est encore plus prononcé dans le cas de HLD dont la durée dépasse souvent la seconde et altère l’intégration d’autres signaux arrivants simultanément. Donc on croit que les effets de HLD se traduisent au niveau du réseaux neuronal du GD comme une composante cruciale pour le codage parcimonieux. En effet, ce type de codage semble être la marque distinctive de cette région étant donné que nous avons aussi observé un faible niveau d’activation chez les interneurones. Cependant, le manque d’activité accrue du réseau inhibiteur antérograde peut être compensé par le maintien d’un gradient GABAergique constant à travers le GD via l’alternance des trois modes de décharges des interneurones. En conclusion, il semble que le codage parcimonieux dans le GD peut être préservé même en absence d’activité soutenue du réseau inhibiteur antérograde et ceci grâce à des mécanismes alternatives d’inhibition prolongée à court terme.The hippocampus is implicated in spatial navigation, the generation and recall of memories, as well as their spatio-temporal organization. These functions are supported by the processes of pattern separation that occurs in the dentate gyrus (DG). Situated at the entry of the hippocampal formation, the DG is well placed to detect and sort novelty patterns amongst the high-density excitatory signals that arrive via the entorhinal cortex (EC). A hallmark of the DG is sparse encoding that is enabled by a combination of low intrinsic excitability of the principal cells and local inhibition. Feedforward inhibition (FFI) is recruited directly by the EC and simultaneously with the granule cells (GCs). Therefore, FFI provides fast GABA release and shapes input integration at the millisecond time scale. This thesis aimed to investigate the interplay of entorhinal excitatory signals with GCs and interneurons, from the FFI in the DG, in the framework of sparse encoding and GC’s characteristic burst firing. We addressed the long-range excitation – local inhibitory network interactions using electrophysiological recordings of GCs – while applying an electrical or optogenetic stimulation of the perforant path (PP) in the DG. We discovered and described a novel delayed-onset inhibitory post synaptic potential (IPSP) in GCs, following PP stimulation in the gamma frequency range. Most importantly, the IPSP was characterized by a large amplitude and prolonged decay, outlasting previously described slow inhibitory events in GCs. The long-lasting hyperpolarization (LLH) caused by the slow IPSPs generates a low excitability time window, alters the GCs firing pattern, and interferes with other stimuli that arrive simultaneously. FFI is therefore a key player in the computational processes that occurs in the DG. However, while many studies have been dedicated to the description of the various types of the interneurons from the FFI, the question of how these cells are synaptically recruited by the EC remains not entirely elucidated. We tackled this problem by recording from interneurons in the DG molecular layer during PP-specific optogenetic stimulation. Light-driven activation of the EC terminals enabled a purely synaptic recruitment of interneurons via endogenous glutamate release. We found that this method of stimulation recruits only a subset of interneurons. In addition, the total number of action potentials (AP) was surprisingly low even at high frequency stimulation. This result is counterintuitive, as strong and persistent inhibitory signals are assumed to restrict GC v activation and maintain sparseness. However, amongst the early firing interneurons, late and regular spiking patterns were clearly distinguishable. Interestingly, some interneurons expressed LLH similar to the GCs, arguing that it could be a commonly used mechanism for regulation of excitability across the hippocampal network. In summary, we show that slow inhibition can result in a prolonged hyperpolarization that significantly alters concurrent input’s integration. We believe that these interactions contribute to important computational processes such as sparse encoding. Interestingly, sparseness seems to be the hallmark of the DG, as we observed a rather low activation of the interneuron network as well. However, the alternating firing of ML-INs could compensate the lack of persistent activity by the continuous GABA release across the DG. Taken together these results offer an insight into a mechanism of feedforward inhibition serving as a sparse neural code generator in the DG

Hippocampus and Human Disease

This chapter focuses on two disorders in which the role of the hippocampus has been extensively investigated: Alzheimer's disease and temporal lobe epilepsy. Although in Alzheimer's disease the disease eventually results in widespread destruction of the cerebral cortex, the damage in the earliest stages of disease is restricted to the entorhinal cortex and the hippocampus, and the memory impairment that results from this disruption of the hippocampal formation represents one of the common characteristics of early onset Alzheimer's disease. In temporal lobe epilepsy, the pathological damage is often restricted to the hippocampus in the form of hippocampal sclerosis. However, unlike Alzheimer's disease, in which the hippocampal damage is secondary to the underlying pathological process, the hippocampus in temporal lobe epilepsy is not only sensitive to damage by seizure activity but can also act as the substrate for epileptic seizure generation

Advances in Neural Signal Processing

Neural signal processing is a specialized area of signal processing aimed at extracting information or decoding intent from neural signals recorded from the central or peripheral nervous system. This has significant applications in the areas of neuroscience and neural engineering. These applications are famously known in the area of brain–machine interfaces. This book presents recent advances in this flourishing field of neural signal processing with demonstrative applications

Advances in Neural Signal Processing

Neural signal processing is a specialized area of signal processing aimed at extracting information or decoding intent from neural signals recorded from the central or peripheral nervous system. This has significant applications in the areas of neuroscience and neural engineering. These applications are famously known in the area of brain–machine interfaces. This book presents recent advances in this flourishing field of neural signal processing with demonstrative applications

Two photon interrogation of hippocampal subregions CA1 and CA3 during spatial behaviour

The hippocampus is crucial for spatial navigation and episodic memory formation. Hippocampal place cells exhibit spatially selective activity within an environment and form the neural basis of a cognitive map of space which supports these mnemonic functions. Hebb’s (1949) postulate regarding the creation of cell assemblies is seen as the pre-eminent model of learning in neural systems. Investigating changes to the hippocampal representation of space during an animal’s exploration of its environment provides an opportunity to observe Hebbian learning at the population and single cell level. When exploring new environments animals form spatial memories that are updated with experience and retrieved upon re-exposure to the same environment, but how this is achieved by different subnetworks in hippocampal CA1 and CA3, and how these circuits encode distinct memories of similar objects and events remains unclear. To test these ideas, we developed an experimental strategy and detailed protocols for simultaneously recording from CA1 and CA3 populations with 2P imaging. We also developed a novel all-optical protocol to simultaneously activate and record from ensembles of CA3 neurons. We used these approaches to show that targeted activation of CA3 neurons results in an increasing excitatory amplification seen only in CA3 cells when stimulating other CA3 cells, and not in CA1, perhaps reflecting the greater number of recurrent connections in CA3. To probe hippocampal spatial representations, we titrated input to the network by morphing VR environments during spatial navigation to assess the local CA3 as well as downstream CA1 responses. To this end, we found CA1 and CA3 neural population responses behave nonlinearly, consistent with attractor dynamics associated with the two stored representations. We interpret our findings as supporting classic theories of Hebbian learning and as the beginning of uncovering the relationship between hippocampal neural circuit activity and the computations implemented by their dynamics. Establishing this relationship is paramount to demystifying the neural underpinnings of cognition