Bursts of beta oscillations across the brain as a neurophysiological correlate of contextual novelty

The retrosplenial cortex and hippocampus are brain regions which have been shown to be highly involved in contextual memory. In order to discover neurophysiological correlates of contextual memory in these regions, we used in vivo electrophysiology in awake, behaving mice while they explored a series of novel and familiar environments. Additionally, in order to better understand the specific neurophysiological effects of Alzheimer’s disease-associated amyloid pathology on the retrosplenial cortex and hippocampus, we compared network activity between wild-type mice and J20 mice, a transgenic mouse model which develops widespread age-related amyloid pathology and memory impairments. We detected transient bursts of beta oscillations in both the retrosplenial cortex and hippocampus that were synchronous between these regions and upregulated during contextual novelty. Moreover, spiking of neurons in the retrosplenial cortex was significantly increased during beta bursts. In J20 mice, we noted numerous examples of altered network activity, including aberrant beta bursting which is not coupled to neuronal spiking. Through the use of EEG recordings in mice, we demonstrated that beta bursts can be detected across the cortex, and are highly synchronous between different brain regions. Finally, we demonstrated that it is possible to pharmacologically induce beta bursting in the retrosplenial cortex in vitro through the use of carbachol, a muscarinic acetylcholine receptor agonist, providing an assay for better understanding the mechanisms underlying beta bursting. These findings suggest that transient beta bursting across the brain provides brief windows of effective communication between brain regions, which may underlie the formation of cortical representation of contexts, and may be impaired in Alzheimer’s disease

Parvalbumin expression and gamma oscillation occurrence increase over time in a neurodevelopmental model of NMDA receptor dysfunction

Dysfunction of the N-methyl-d-aspartate receptor (NMDAR) is thought to play a role in the pathophysiology of neurodevelopmental diseases like schizophrenia. To study the effects of NMDAR dysfunction on synaptic transmission and network oscillations, we used hippocampal tissue of NMDAR subunit GluN2A knockout (KO) mice. Field excitatory postsynaptic potentials were recorded in acute hippocampal slices of adult animals. Synaptic transmission was impaired in GluN2A KO slices compared to wild-type (WT) slices. Further, to investigate whether NMDAR dysfunction would alter neurodevelopment in vitro, we used organotypic hippocampal slice cultures of WT and GluN2A KO mice. Immunostaining performed with cultures kept two, seven, 14, 25 days in vitro (DIV) revealed an increasing expression of parvalbumin (PV) over time. As a functional readout, oscillatory activity induced by the cholinergic agonist carbachol was recorded in cultures kept seven, 13, and 26 DIV using microelectrode arrays. Initial analysis focused on the occurrence of delta, theta, beta and gamma oscillations over genotype, DIV and hippocampal area (CA1, CA3, dentate gyrus (DG)). In a follow-up analysis, we studied the peak frequency and the peak power of each of the four oscillation bands per condition. The occurrence of gamma oscillations displayed an increase by DIV similar to the PV immunostaining. Unlike gamma occurrence, delta, theta, and beta occurrence did not change over time in culture. The peak frequency and peak power in the different bands of the oscillations were not different in slices of WT and GluN2A KO mice. However, the level of PV expression was lower in GluN2A KO compared to WT mice. Given the role of PV-containing fast-spiking basket cells in generation of oscillations and the decreased PV expression in subjects with schizophrenia, the study of gamma oscillations in organotypic hippocampal slices represents a potentially valuable tool for the characterization of novel therapeutic drugs

Astrocytic modulation of neuronal network oscillations

The synchronization of the neuron’s membrane potential results in the emergence of neuronal oscillations at multiple frequencies that serve distinct physiological functions (e.g. facilitation of synaptic plasticity) and correlate with different behavioural states (e.g. sleep, wakefulness, attention). It has been postulated that at least ten distinct mechanisms are required to cover the large frequency range of neuronal oscillations in the cortex, including variations in the concentration of extracellular neurotransmitters and ions, as well as changes in cellular excitability. However, the mechanism that gears the transition between different oscillatory frequencies is still unknown. Over the past decade, astrocytes have been the focus of much research, mainly due to (1) their close association with synapses forming what is known today as the “tripartite synapse”, which allows them to bidirectionally interact with neurons and modulate synaptic transmission; (2) their syncytium-like activity, as they are electrically coupled via gap junctions and actively communicate through Ca2+ waves; and (3) their ability to regulate neuronal excitability via glutamate uptake and tight control of the extracellular K+ levels via a process termed K+ clearance. In this thesis we hypothesized that astrocytes, in addition to their role as modulators of neuronal excitability, also act as “network managers” that can modulate the overall network oscillatory activity within their spatial domain. To do so, it is proposed that astrocytes fine-tune their K+ clearance capabilities to affect neuronal intrinsic excitability properties and synchronization with other neurons, thus mediating the transitions between neuronal network oscillations at different frequencies. To validate or reject this hypothesis I have investigated the potential role of astrocytes in modulating cortical oscillations at both cellular and network levels, aiming at answering three main research questions: a) what is the impact of alterations in astrocytic K+ clearance mechanisms on cortical networks oscillatory dynamics? b) what specific neuronal properties underlying the generation of neuronal oscillations are affected as a result of impairments in the astrocytic K+ clearance process? and c) what are the bidirectional mechanisms between neurons and astrocytes (i.e. neuromodulators) that specifically affect the K+ clearance process to modulate the network activity output? In the first experimental chapter I used electrophysiological recordings and pharmacological manipulations to dissect the contribution of the different astrocytic K+ clearance mechanisms to the modulation of neuronal network oscillations at multiple frequencies. A key finding was that alterations in membrane properties of layer V pyramidal neurons strongly correlated with the network behaviour following impairments in astrocytic K+ clearance capabilities, depicted as enhanced excitability underlying the amplification of high-frequency oscillations, especially within the beta and gamma range. The second experimental chapter describes a combinatorial approach based on K+-selective microelectrode recordings and optical imaging of K+ ions used to quantitatively determine extracellular K+ changes and to follow the spatiotemporal distribution of K+ ions under both physiological and altered K+ clearance conditions, which affected the K+ clearance rate. The impact of different neuromodulators on astrocytic function is discussed in the third experimental chapter. Using extracellular K+ recordings and Ca2+ imaging I found that some neuromodulators act specifically on astrocytic receptors to affect both K+ clearance mechanisms and Ca2+ signalling, as evidenced by reduced K+ clearance rates and altered evoked Ca2+ signals. Overall, this thesis provides new insights regarding the impact of astrocytic K+ clearance mechanisms on modulating neuronal properties at both cellular and network levels, which in turn imposes alterations on neuronal oscillations that are associated with different behavioural states

Phase-amplitude coupled persistent theta and gamma oscillations in rat primary motor cortex in vitro

In vivo, theta (4-7 Hz) and gamma (30-80 Hz) neuronal network oscillations are known to coexist and display phase-amplitude coupling (PAC). However, in vitro, these oscillations have for many years been studied in isolation. Using an improved brain slice preparation technique we have, using co-application of carbachol (10 μM) and kainic acid (150 nM), elicited simultaneous theta (6.6 ± 0.1 Hz) and gamma (36.6 ± 0.4 Hz) oscillations in rodent primary motor cortex (M1). Each oscillation showed greatest power in layer V. Using a variety of time series analyses we detected significant cross-frequency coupling 74% of slice preparations. Differences were observed in the pharmacological profile of each oscillation. Thus, gamma oscillations were reduced by the GABAA receptor antagonists, gabazine (250 nM and 2 μM), and picrotoxin (50 μM) and augmented by AMPA receptor antagonism with SYM2206 (20 μM). In contrast, theta oscillatory power was increased by gabazine, picrotoxin and SYM2206. GABAB receptor blockade with CGP55845 (5 μM) increased both theta and gamma power, and similar effects were seen with diazepam, zolpidem, MK801 and a series of metabotropic glutamate receptor antagonists. Oscillatory activity at both frequencies was reduced by the gap junction blocker carbenoxolone (200 μM) and by atropine (5 μM). These data show theta and gamma oscillations in layer V of rat M1 in vitro are cross-frequency coupled, and are mechanistically distinct. The development of an in vitro model of phase-amplitude coupled oscillations will facilitate further mechanistic investigation of the generation and modulation of coupled activity in mammalian cortex

Are Different Rhythms Good for Different Functions?

This essay discusses the relationship between the physiology of rhythms and potential functional roles. We focus on how the biophysics underlying different rhythms can give rise to different abilities of a network to form and manipulate cell assemblies. We also discuss how changes in the modulatory setting of the rhythms can change the flow of information through cortical circuits, again tying physiology to computation. We suggest that diverse rhythms, or variations of a rhythm, can support different components of a cognitive act, with multiple rhythms potentially playing multiple roles

Direct and indirect cholinergic septo-hippocampal pathways cooperate to structure spiking activity in the hippocampus

The medial septum/vertical diagonal band of Broca complex (MSvDB) is a key structure that modulates hippocampal rhythmogenesis. Cholinergic neurons of the MSvDB play a central role in generating and pacing theta-band oscillations in the hippocampal formation during exploration, novelty detection, and memory encoding. However, how precisely cholinergic neurons affect hippocampal oscillatory activity and spiking rates of hippocampal neurons in vivo, has remained elusive. I therefore used silicon probe recordings of local field potentials and unit activity in the dorsal hippocampus in combination with cell type specific optogenetic activation of cholinergic MSvDB neurons to study the effects of synaptically released acetylcholine on hippocampal network activity in urethane-anesthetized mice.In vivo optogenetic activation of cholinergic MSvDB neurons induced hippocampal rhythmogenesis at the theta (3-6 Hz) and slow gamma (26-48 Hz) frequency range with a suppression of peri-theta frequencies. Interestingly, this effect was independent from the stimulation frequency. In addition, stimulation of cholinergic MSvDB neurons resulted in a net increase of interneuron firing with a concomitant net decrease of principal cell firing in the hippocampal CA3 subfield. I used focal injections of cholinergic blockers either into the MSvDB or the hippocampus to demonstrate that cholinergic MSvDB neurons modulate hippocampal network activity via two distinct pathways. Focal injection of a cholinergic blocker cocktail into the hippocampus strongly diminished the cholinergic stimulation-induced spiking rate modulation of hippocampal interneurons and principal cells. This demonstrates that modulation of neuronal activity in hippocampal subfield CA3 by cholinergic MSvDB neurons is mediated via direct septo-hippocampal projections. In contrast, focal injection of atropine, a blocker of the muscarinic type of acetylcholine receptors, into the MSvDB had no effect on spiking rate modulation in CA3, but abolished hippocampal theta synchronization. This strongly suggests that activity of an indirect septo-hippocampal pathway induces hippocampal theta oscillations via an intraseptal relay. Furthermore, cholinergic neurons depolarized parvalbumin-positive (PV+) GABAergic neurons within the MSvDB in vitro, and optogenetic activation of these fast spiking neurons in vivo induced hippocampal rhythmic activity precisely at the stimulation frequency. Taken together, these data suggest an intraseptal relay with a strong contribution of PV+ GABAergic MSvDB neurons in pacing hippocampal theta oscillations. Activation of both the direct and indirect pathways causes a reduction in CA3 pyramidal neuron firing and a more precise coupling to theta oscillatory phase with CA3 interneurons preferentially firing at the descending phase and CA3 principal neurons preferentially firing near the trough of the ongoing theta oscillation recorded at the pyramidal cell layer. The two identified anatomically and functionally distinct pathways are likely relevant for cholinergic control of encoding vs. retrieval modes in the hippocampus

In vitro neuronal cultures on MEA: an engineering approach to study physiological and pathological brain networks

Reti neuronali accoppiate a matrici di microelettrodi: un metodo ingegneristico per studiare reti cerebrali in situazioni fisiologiche e patologich

Making Waves in the Brain: What Are Oscillations, and Why Modulating Them Makes Sense for Brain Injury.

Traumatic brain injury (TBI) can result in persistent cognitive, behavioral and emotional deficits. However, the vast majority of patients are not chronically hospitalized; rather they have to manage their disabilities once they are discharged to home. Promoting recovery to pre-injury level is important from a patient care as well as a societal perspective. Electrical neuromodulation is one approach that has shown promise in alleviating symptoms associated with neurological disorders such as in Parkinson's disease (PD) and epilepsy. Consistent with this perspective, both animal and clinical studies have revealed that TBI alters physiological oscillatory rhythms. More recently several studies demonstrated that low frequency stimulation improves cognitive outcome in models of TBI. Specifically, stimulation of the septohippocampal circuit in the theta frequency entrained oscillations and improved spatial learning following TBI. In order to evaluate the potential of electrical deep brain stimulation for clinical translation we review the basic neurophysiology of oscillations, their role in cognition and how they are changed post-TBI. Furthermore, we highlight several factors for future pre-clinical and clinical studies to consider, with the hope that it will promote a hypothesis driven approach to subsequent experimental designs and ultimately successful translation to improve outcome in patients with TBI

A neurophysiological and proteomic study of cognitive enhancing strategies

Improving cognitive function is a growing area of interest for pharmaceutical companies. With an ageing population, cognitive decline is becoming a greater problem. Understanding the physiological effects of nootropic drugs and the changes that occur during cognitive enhancement will enable the design of safer treatments to enhance cognition. In this thesis cognitive enhancing strategies are investigated using neurophysiological and proteomic approaches. The effects of two classes of putatively cognitive enhancing drugs on emergent network oscillatory activities in hippocampal slices are investigated. The effect of an enriched environment, which causes an improvement in cognitive function, on the expression level of proteins in the hippocampus is also investigated. The development of a new muscarinic acetylcholine receptor (mAChR) agonist that is selective for the M1 mAChR subtype, called 77-LH-281, has recently been achieved. 77-LH-281 binds to an allosteric site of the M1 mAChR which accounts for its increased selectivity over other mAChR agonists. This agonist causes gamma frequency oscillatory activity in hippocampal slices, a pattern of network activity that the in vivo equivalent of which is associated with cognitive processes. This gamma activity is dependent upon both excitatory and inhibitory networks. 77-LH-281 does not promote epileptiform-like activity in naïve slices as well as a range of models of epileptiform activity, unlike non-subtype selective mAChR agonists like oxotremorine-M. Oxotremorine-M changes the slow inter-ictal-like events following application of 4-AP and NBQX into continuous beta frequency oscillations. This action is not mediated by the M1 mAChR. Thus selective M¬1 mAChR display a preferable range of oscillatory activities compared to non-subtype selective mAChR agonists. Ampakines are a further class of nootropic drugs. Ampakines are positive modulators of AMPA-type glutamate receptors and they cause improvements in cognitive function of laboratory animals and humans. The ampakines investigated in this thesis are CX691, which increases the amplitude of currents through the AMPA receptor, and CX546, which increases the length of time the AMPA receptor is open. These ampakines do not induce oscillatory activity in naïve hippocampal slices, but they increase the frequency of inter-ictal-like epileptiform activity. CX546 also induces ictal-like activity in the 4-AP induced epileptiform event model. Ampakines may therefore promote epileptiform activity in individuals that are susceptible to epilepsy. Exposure to an enriched environment leads to improvements in cognitive performance. This behavioural change is mediated by changes at the level of the proteome. Exposure to an enriched environment changes the expression of many classes of proteins including signalling proteins and proteins that are involved in the structural changes that occur during cognitive enhancement. One of the proteins that significantly changes in expression is a protein that is associated with cognitive deficits, known as MeCP2. MeCP2 is a transcriptional repressor and increases in expression in the enriched environment. This thesis demonstrates the diversity of molecular, cellular and network level approaches that can be used to induce and investigate cognitive enhancement. A combination of these approaches enables the in vitro evaluation of current cognitive enhancing strategies and may lead to the the development of novel approaches to enhance cognitive function