On the role of parvalbumin interneurons in neuronal network activity in the prefrontal cortex

The prefrontal cortex (PFC) is an area important for executive functions, the initiation and temporal organization of goal-directed behavior, as well as social behaviors. Inhibitory interneurons expressing parvalbumin (PV) have a vital role in modulating PFC circuit plasticity and output, as inhibition by PV interneurons on excitatory pyramidal neurons regulates the excitability of the network. Thus, dysfunctions of prefrontal PV interneurons are implicated in the pathophysiology of a range of PFC-dependent neuropsychiatric disorders characterized by excitation and inhibition (E/I) imbalance and impaired gamma oscillations. In particular, the hypofunction of receptors important for neurotransmission and regulating cellular functions, such as the N-methyl-D-aspartate receptors (NMDARs) and the tyrosine receptor kinase B (trkB), has been implicated in PV dysfunction. Notably, this hypofunction is known to impair the normal development of PV interneurons. However, it can also affect adult brain activity. The effects of altered receptors on PV interneurons are multiple, from impaired morphological connectivity to disruption of intrinsic activity, but have not yet been fully characterized. Moreover, the effects of deficits of PV neuron-mediated inhibition on neuronal network activity are complex, involved with compensatory mechanisms, and not fully understood either. For instance, the E/I imbalance due to PV inhibition has been suggested to functionally disrupt the cortex, which can be observed through an abnormal increase in broadband gamma activity. But as the synchronous activity of cortical PV interneurons is necessary for the generation of cortical gamma oscillations, it is paradoxical that deficient PV inhibition is associated with increased broadband gamma power. This thesis aims to examine the role of PV interneurons in shaping neuronal network activity in the mouse PFC by investigating the microscopic to macroscopic functional effects of disrupting receptors necessary for the proper activity of PV interneurons. In paper I, we observed that the increase of broadband gamma power due to NMDAR hypofunction in PV neurons is associated with asynchronies of network activity, confirming that dysfunction of neuronal inhibition can cause desynchronization at multiple time scales (affecting entrainment of spikes by the LFP, as well as cross-frequency coupling and brain states fragmentation). In Paper II, we prompted and analyzed the rippling effect of PV dysfunction in the adult PFC by expressing a dominant-negative trkB receptor specifically in PV interneurons. Despite avoiding interfering with the development of the brain, we found pronounced morphological and functional alterations in the targeted PV interneurons. These changes were associated with unusual aggressive behavior coupled with gamma-band alterations and a decreased modulation of prefrontal excitatory neuronal populations by PV interneurons. Thus, the work presented in this thesis furthers our understanding of the role of PV function in PFC circuitry, particularly of two receptors that are central to the role of PV interneurons in coordinating local circuit activity. A better understanding of the potential mechanisms that could explain the neuronal changes seen in individuals with neuropsychiatric dysfunctions could lead to using gamma oscillations or BDNF-trkB levels as biomarkers in psychiatric disorders. It also presents possibilities for potential treatments designed around reestablishing E/I balance by modifying receptor levels in particular cell types

Network asynchrony underlying increased broadband gamma power

Synchronous activity of cortical inhibitory interneurons expressing parvalbumin (PV) underlies expression of cortical γ rhythms. Paradoxically, deficient PV inhibition is associated with increased broadband γ power in the local field potential. Increased baseline broadband γ is also a prominent characteristic in schizophrenia and a hallmark of network alterations induced by NMDAR antagonists, such as ketamine. Whether enhanced broadband γ is a true rhythm, and if so, whether rhythmic PV inhibition is involved or not, is debated. Asynchronous and increased firing activities are thought to contribute to broadband power increases spanning the γ band. Using male and female mice lacking NMDAR activity specifically in PV neurons to model deficient PV inhibition, we here show that neuronal activity with decreased synchronicity is associated with increased prefrontal broadband γ power. Specifically, reduced spike time precision and spectral leakage of spiking activity because of higher firing rates (spike “contamination”) affect the broadband γ band. Desynchronization was evident at multiple time scales, with reduced spike entrainment to the local field potential, reduced cross-frequency coupling, and frag- mentation of brain states. Local application of S(1)-ketamine in (control) mice with intact NMDAR activity in PV neurons triggered network desynchronization and enhanced broadband γ power. However, our investigations suggest that disparate mechanisms underlie increased broadband γ power caused by genetic alteration of PV interneurons and ketamine-induced power increases in broadband c. Our study confirms that enhanced broadband γ power can arise from asynchronous activ- ities and demonstrates that long-term deficiency of PV inhibition can be a contributor.ERCSTINT Program Joint Brazilian-Swedish Research Collaboration GrantCAPES-STINT Program GrantKnut and Alice Wallenberg FoundationSwedish Research CouncilKarolinska InstitutetAccepte

The Opposing Roles of GluN2C and GluN2D NMDA Receptor Subunits in Modulating Neuronal Oscillations

N-methyl-D-aspartate receptors (NMDARs) are ligand-gated ion channels consisting of two GluN1 subunits and two other subunits from among GluN2A-2D and GluN3A-3B subunits. NMDARs play critical roles in synaptic plasticity, learning and memory, and higher brain function such as cognition and perception. Dysfunction of NMDARs (hyper-function and hypo-function of NMDARs) are related to various diseases, including stroke, schizophrenia, Alzheimer’s disease, and others. However, to date, NMDARs antagonists have mostly failed in clinical trials due to adverse effects. NMDARs antagonists replicate the core symptoms of schizophrenia which may underlie its ability to alter neuronal oscillations in the neural circuitry of different brain regions. Recent evidence has shown that GluN2C subunits of NMDAR are expressed in astrocytes in the cortex, and that GluN2D NMDAR subunits are enriched in the parvalbumin-containing GABAergic inhibitory interneurons in the cortex and midbrain structures. Other studies have shown that both astrocytes and parvalbumin-containing interneurons play an essential role in generating and maintaining neuronal oscillations. These findings imply that GluN2C and GluN2D subunits may be involved in the distinct neural circuitry which regulates neuronal oscillations and thus influence the brain function and contribute to various diseases states. The initial aims of this dissertation are to determine if GluN2C and GluN2D subunits have a role in regulating neuronal oscillations. We also measured the auditory evoked responses in wildtype and GluN2C- and GluN2D-KO mice. Lastly, we use ketamine as the tool drug to determine the role of NMDARs in neuronal oscillations in a CDKL5-KO mouse model. We found that spontaneous basal neuronal oscillations were elevated in GluN2C- and GluN2D-KO mice compared to WT mice. NMDARs antagonists increased the power of neuronal oscillations in WT mice; we found drug-induced power increase is abolished in GluN2D-KO mice and is augmented in GluN2C-KO mice. Furthermore, we also found GluN2D-KO mice displayed abnormal auditory evoked responses. Lastly, we test subunit-selective NMDARs drug and NMDARs allosteric modulators with distinct subunits selectivity developed by our lab, including PAMs and NAMs on these KO models

Shifted phase of EEG cross-frequency coupling in individuals with Phelan-McDermid syndrome

Background Phelan-McDermid Syndrome (PMS) is a rare condition caused by deletion or mutation of the SHANK3 gene. Individuals with PMS frequently present with intellectual disability, autism spectrum disorder, and other neurodevelopmental challenges. Electroencephalography (EEG) can provide a window into network-level function in PMS. Methods Here, we analyze EEG data collected across multiple sites in individuals with PMS (n = 26) and typically developing individuals (n = 15). We quantify oscillatory power, alpha-gamma phase-amplitude coupling strength, and phase bias, a measure of the phase of cross frequency coupling thought to reflect the balance of feedforward (bottom-up) and feedback (top-down) activity. Results We find individuals with PMS display increased alpha-gamma phase bias (U = 3.841, p < 0.0005), predominantly over posterior electrodes. Most individuals with PMS demonstrate positive overall phase bias while most typically developing individuals demonstrate negative overall phase bias. Among individuals with PMS, strength of alpha-gamma phase-amplitude coupling was associated with Sameness, Ritualistic, and Compulsive behaviors as measured by the Repetitive Behavior Scales-Revised (Beta = 0.545, p = 0.011). Conclusions Increased phase bias suggests potential circuit-level mechanisms underlying phenotype in PMS, offering opportunities for back-translation of findings into animal models and targeting in clinical trials

Ion channels in EEG: isolating channel dysfunction in NMDA receptor antibody encephalitis

Neurological and psychiatric practice frequently lack diagnostic probes that can assess mechanisms of neuronal communication non-invasively in humans. In N-methyl-D-aspartate (NMDA) receptor antibody encephalitis, functional molecular assays are particularly important given the presence of NMDA antibodies in healthy populations, the multifarious symptomology and the lack of radiological signs. Recent advances in biophysical modelling techniques suggest that inferring cellular-level properties of neural circuits from macroscopic measures of brain activity is possible. Here, we estimated receptor function from EEG in patients with NMDA receptor antibody encephalitis (n = 29) as well as from encephalopathic and neurological patient controls (n = 36). We show that the autoimmune patients exhibit distinct fronto-parietal network changes from which ion channel estimates can be obtained using a microcircuit model. Specifically, a dynamic causal model of EEG data applied to spontaneous brain responses identifies a selective deficit in signalling at NMDA receptors in patients with NMDA receptor antibody encephalitis but not at other ionotropic receptors. Moreover, though these changes are observed across brain regions, these effects predominate at the NMDA receptors of excitatory neurons rather than at inhibitory interneurons. Given that EEG is a ubiquitously available clinical method, our findings suggest a unique re-purposing of EEG data as an assay of brain network dysfunction at the molecular level

Mechanistic insights into neuronal oscillatory activity in the dopamine-intact and dopamine-depleted primary motor cortex

In Parkinson’s disease (PD) the loss of the neurotransmitter dopamine (DA) results in abnormal oscillations of the cortico-basal ganglia network, the emergence of which correlate with symptoms. Increased oscillatory power in the primary motor cortex (M1) is reduced by dopamine replacement therapy and by targeted stimulation, suggesting that M1 plays an important role in the pathology of PD. In this study we have investigated, using pharmacology, the mechanisms by which oscillatory activity in rat M1 is generated and determined the power changes associated with DA depletion and DA receptor modulation. Extracellular local field potential recordings were made in brain slices of M1 which were prepared using a modified protocol to improve viability. Co-application of carbachol (5 μM) and kainic acid (100 nM) elicited simultaneous theta (4-8 Hz) and gamma (30-40 Hz) oscillations in layer V of M1. These oscillations displayed phase-amplitude coupling; the first report of such findings in vitro. These oscillations were found to be pharmacologically distinct with theta oscillations generated by intrinsic non-synaptic mechanisms while gamma oscillations required contributing excitatory and inhibitory networks. Following successful unilateral lesions using 6-hydroxydopamine (6-OHDA), as determined by the adjusting step test, DA-depleted (ipsilateral) and DA-intact (contralateral) slices were obtained. Although no difference in the oscillatory profile of M1 ipsilateral, contralateral or age-matched control (AMC) slices was found, bath application of DA reduced gamma power only in the ipsilateral slices and amphetamine only decreased gamma power in contralateral slices. Furthermore, D2-like receptor activation consistently increased both theta and gamma power in contralateral and AMC slices, while only theta power was increased in ipsilateral slices. Overall, these data suggest that DA, through action at multiple sites, differentially modulates the power of both theta and gamma oscillations in M1. Using the 6-OHDA model, the oscillatory activity of M1 in vivo was investigated. Successful lesions were determined by using the rotometer, the locomotor activity and the adjusting stepping tests at 2-4 weeks post-surgery. Further testing at 22 weeks post-surgery indicated the long-term stability of the lesions. Using depth electrode and EEG recordings, oscillatory activity in the 2-10 Hz range was found in the ipsilateral and contralateral hemispheres of both lesioned and sham animals. However, only in the ipsilateral hemisphere of DA-depleted animals did we detect a 30-40 Hz oscillatory peak, which was localised to layer V of M1. In EEG recordings this led to a significant increase in the interhemispheric ratio. Using depth electrode recordings, the ipsilateral 30-40 Hz oscillation (but not 2-10 Hz oscillation) was reduced by the administration of L-DOPA (6 mg/kg) with a reduction in interhemispheric ratio. However, administration of zolpidem (0.3 mg/kg), which previously reduced abnormal beta oscillatory activity in vivo and in vitro resulting in the rebalancing of interhemispheric beta power (Hall et al., 2014; Prokic et al., 2015), was without effect. Overall, these studies demonstrate that M1 alone can generate multiple, pharmacologically distinct, but interacting oscillations, which contribute to pathological activity in the DA-depleted state

Generation and analysis of transgenic mice expressing CRE recombinase in defined interneurons

GABAergic interneurons are the main source of inhibition in the central nervous system. In addition they play a crucial role during development since in a paradoxical fashion they are the origin of the first excitatory signals in the immature brain. GABAergic interneurons comprise about 10 – 20% of the neuronal cell population and they can be divided into several subtypes. GABAergic interneuron classifications have been based on different criteria, including anatomical, neurochemical or physiological characteristics. Although the overall number of interneurons is small compared to that of principal cells, by virtue of their connectivity, interneurons are able to shape and regulate the activity of numerous principal cells and thus influence network activity. To elucidate the role of interneurons during development and in the mature brain specific modifications of their molecular and physiological properties are required and can be achieved by selective ablation of distinct genes. An established and widely used technique is that of the CRE/loxP system. For ablation of a desired gene in a cell-type specific fashion the generation of mice expressing CRE recombinase in a subset of cells plus the generation of mice with a floxed allele are a prerequisite. The aim of this study was the generation of mice with CRE expression in all GABAergic interneurons and of mice with restricted expression of CRE recombinase in a subset of GABAergic interneurons, the somatostatin-positive interneurons. To this end mice with CRE expression under the control of the GAD67 promoter (GAD67CRE/+) - a common feature of almost all interneurons - and mice with CRE recombinase expression under the control of the somatostatin promoter (SOMCRE/+) were generated. Immunohistochemical analysis of both GAD67CRE/+ and SOMCRE/+ mice provided evidence that CRE recombinase is functional in vivo. Co-localisation studies of CRE recombinase and endogenous GAD67 expression, demonstrated a 100% overlap. Double-labelling experiments of endogenous somatostatin and CRE recombinase demonstrated a good correspondence in the hippocampus but less so in other brain regions. No developmental or behavioural deficits were observed as a consequence of the genetic manipulations. Cell-type specific ablations of several genes of interest, e.g. trkB receptors in GABAergic interneurons, NR1 and GluR-A subunits in somatostatin-positive interneurons are currently being generated and will help provide more insights into the function of GABAergic interneurons during development and their involvement in specific network activity

From mechanisms to markers: novel noninvasive EEG proxy markers of the neural excitation and inhibition system in humans.

Brain function is a product of the balance between excitatory and inhibitory (E/I) brain activity. Variation in the regulation of this activity is thought to give rise to normal variation in human traits, and disruptions are thought to potentially underlie a spectrum of neuropsychiatric conditions (e.g., Autism, Schizophrenia, Downs' Syndrome, intellectual disability). Hypotheses related to E/I dysfunction have the potential to provide cross-diagnostic explanations and to combine genetic and neurological evidence that exists within and between psychiatric conditions. However, the hypothesis has been difficult to test because: (1) it lacks specificity-an E/I dysfunction could pertain to any level in the neural system- neurotransmitters, single neurons/receptors, local networks of neurons, or global brain balance - most researchers do not define the level at which they are examining E/I function; (2) We lack validated methods for assessing E/I function at any of these neural levels in humans. As a result, it has not been possible to reliably or robustly test the E/I hypothesis of psychiatric disorders in a large cohort or longitudinal patient studies. Currently available, in vivo markers of E/I in humans either carry significant risks (e.g., deep brain electrode recordings or using Positron Emission Tomography (PET) with radioactive tracers) and/or are highly restrictive (e.g., limited spatial extent for Transcranial Magnetic Stimulation (TMS) and Magnetic Resonance Spectroscopy (MRS). More recently, a range of novel Electroencephalography (EEG) features has been described, which could serve as proxy markers for E/I at a given level of inference. Thus, in this perspective review, we survey the theories and experimental evidence underlying 6 novel EEG markers and their biological underpinnings at a specific neural level. These cheap-to-record and scalable proxy markers may offer clinical utility for identifying subgroups within and between diagnostic categories, thus directing more tailored sub-grouping and, therefore, treatment strategies. However, we argue that studies in clinical populations are premature. To maximize the potential of prospective EEG markers, we first need to understand the link between underlying E/I mechanisms and measurement techniques