The Current Status of Somatostatin-Interneurons in Inhibitory Control of Brain Function and Plasticity

The mammalian neocortex contains many distinct inhibitory neuronal populations to balance excitatory neurotransmission. A correct excitation/inhibition equilibrium is crucial for normal brain development, functioning, and controlling lifelong cortical plasticity. Knowledge about how the inhibitory network contributes to brain plasticity however remains incomplete. Somatostatin-(SST-) interneurons constitute a large neocortical subpopulation of interneurons, next to parvalbumin-(PV-) and vasoactive intestinal peptide-(VIP-) interneurons. Unlike the extensively studied PV-interneurons, acknowledged as key components in guiding ocular dominance plasticity, the contribution of SST-interneurons is less understood. Nevertheless, SST-interneurons are ideally situated within cortical networks to integrate unimodal or cross-modal sensory information processing and therefore likely to be important mediators of experience-dependent plasticity. The lack of knowledge on SST-interneurons partially relates to the wide variety of distinct subpopulations present in the sensory neocortex. This review informs on those SST-subpopulations hitherto described based on anatomical, molecular, or electrophysiological characteristics and whose functional roles can be attributed based on specific cortical wiring patterns. A possible role for these subpopulations in experience-dependent plasticity will be discussed, emphasizing on learning-induced plasticity and on unimodal and cross-modal plasticity upon sensory loss. This knowledge will ultimately contribute to guide brain plasticity into well-defined directions to restore sensory function and promote lifelong learning

Therapeutic depletion of CCR8+ tumor-infiltrating regulatory T cells elicits antitumor immunity and synergizes with anti-PD-1 therapy.

BACKGROUND: Modulation and depletion strategies of regulatory T cells (Tregs) constitute valid approaches in antitumor immunotherapy but suffer from severe adverse effects due to their lack of selectivity for the tumor-infiltrating (ti-)Treg population, indicating the need for a ti-Treg specific biomarker. METHODS: We employed single-cell RNA-sequencing in a mouse model of non-small cell lung carcinoma (NSCLC) to obtain a comprehensive overview of the tumor-infiltrating T-cell compartment, with a focus on ti-Treg subpopulations. These findings were validated by flow cytometric analysis of both mouse (LLC-OVA, MC38 and B16-OVA) and human (NSCLC and melanoma) tumor samples. We generated two CCR8-specific nanobodies (Nbs) that recognize distinct epitopes on the CCR8 extracellular domain. These Nbs were formulated as tetravalent Nb-Fc fusion proteins for optimal CCR8 binding and blocking, containing either an antibody-dependent cell-mediated cytotoxicity (ADCC)-deficient or an ADCC-prone Fc region. The therapeutic use of these Nb-Fc fusion proteins was evaluated, either as monotherapy or as combination therapy with anti-programmed cell death protein-1 (anti-PD-1), in both the LLC-OVA and MC38 mouse models. RESULTS: We were able to discern two ti-Treg populations, one of which is characterized by the unique expression of Ccr8 in conjunction with Treg activation markers. Ccr8 is also expressed by dysfunctional CD4+ and CD8+ T cells, but the CCR8 protein was only prominent on the highly activated and strongly T-cell suppressive ti-Treg subpopulation of mouse and human tumors, with no major CCR8-positivity found on peripheral Tregs. CCR8 expression resulted from TCR-mediated Treg triggering in an NF-κB-dependent fashion, but was not essential for the recruitment, activation nor suppressive capacity of these cells. While treatment of tumor-bearing mice with a blocking ADCC-deficient Nb-Fc did not influence tumor growth, ADCC-prone Nb-Fc elicited antitumor immunity and reduced tumor growth in synergy with anti-PD-1 therapy. Importantly, ADCC-prone Nb-Fc specifically depleted ti-Tregs in a natural killer (NK) cell-dependent fashion without affecting peripheral Tregs. CONCLUSIONS: Collectively, our findings highlight the efficacy and safety of targeting CCR8 for the depletion of tumor-promoting ti-Tregs in combination with anti-PD-1 therapy

Dissecting the role of somatostatin-interneurons in visual cortex plasticity of the adult mouse

Neuroplasticity allows the brain to organize and reorganize itself structurally and functionally based on sensory information, a well-studied phenomenon in the mammalian visual cortex. Previously, our research lab observed after a period of seven weeks of unilateral vision loss through monocular enucleation (7wME) in adult mice, that the contralateral visual cortex almost completely reactivated, by measuring the expression of the immediate early gene zif268. In the binocular region, open-eye potentiation serves this reactivation, whereas the monocular zone reactivates partially due to cross-modal recruitment by other intact sensory modalities. A key regulator of these experience-dependent modifications is the balance between excitation and inhibition, yet many distinct inhibitory subtypes populate the cortex, which challenges our understanding of the cellular and molecular basics of cortical plasticity. In this dissertation we aimed to investigate by means of cell type-specific neuromodulation strategies, how a particular inhibitory cell type regulates adult, ME-induced, cortical plasticity. We targeted somatostatin (SST)-interneurons due to their central position within the cortical circuit where they can precisely regulate the integration of incoming signals onto dendritic trees of pyramidal, excitatory neurons. In order to achieve cell type-specific expression of neuromodulatory transgenes in the cell population and cortical area of interest, we first validated a recombinant adeno-associated viral (rAAV) vector approach to transduce neurons within the visual cortex of C57BL/6J mice. Several rAAV serotypes, each resulting in different transduction efficiencies and tropisms, were tested for expression confined to the primary visual cortex, V1. Three promoter-sequences were tested for cell type-specific transgene expression: the cytomegalovirus (CMV) promoter, and two versions (0.4 kb and 1.3 kb) of Ca2+/Calmodulin dependent kinase a (CaMKIIa) promoter. The results indicated that rAAV2/7 resulted in a reproducible, wide expression pattern, confined within V1. CMV resulted in widespread expression in both excitatory and inhibitory cell types, whereas CaMKIIa resulted in expression predominantly in excitatory neurons with the highest excitatory specificity for CaMKIIa 0.4. Next we investigated the contribution of SST-interneuron activity to cortical plasticity, by using the rAAV2/7 construct with a CMV promoter carrying a floxed transgene to achieve cell type-specific neuromodulation in SST-Cre transgenic mice. A stable-step function opsin (SSFO) was used to achieve optogenetic activation upon intracranial light delivery. The mutant G-protein coupled receptor hM4Di, a designer receptor exclusively activated by designer drugs (DREADD), was used to achieve chemogenetic silencing of SST-interneurons upon intraperitoneal injection of the designer drug clozapine N-oxide (CNO). Short-term activation or silencing of SST-interneurons prior to ME, resulted either in a strongly reduced, versus an increased reactivation of visual cortex after 7wME, respectively, as measured by zif268-mRNA expression. Furthermore, we combined these experiments with dark exposure (DE) pretreatment, a strategy known to affect the potential for cross-modal plasticity by altering the excitation/inhibition balance. DE combined with activating SST-interneurons resulted in the most severe lack of reactivation, whereas DE combined with silencing SST-interneurons resulted in a recovery profile comparable to non-perturbed 7wME mice. These results suggest that SST-interneuron activity and DE each address distinct mechanisms both working in the same direction to block reactivation in the ME-affected visual cortex. On the other hand, SST-interneuron silencing overcame the effects of DE, suggesting that even though DE is a non-invasive strategy to modulate the cortical response to sensory loss, directly manipulating the activity of specific neuronal subsets modulates the brain's ability to tap into its plasticity-potential in a more powerful way. These results are a first indication that SST-interneurons are pivotal players in regulating adult cortical plasticity upon a complete, unilateral loss of a sensory modality, which is characterized by cross-modal recruitment via intact sensory modalities. Functional assessment of cross-modal processing in these recovered areas, and neuromodulation of upstream input sources of SST-interneurons, will be required to further elucidate how manipulating the neuronal cortical circuit can switch cross-modal plasticity on or off. In all, the advances in cell type-specific neuromodulation strategies offer new opportunities both for fundamental research as well as clinical therapies to help a damaged or sensory deprived brain to restore its functionality throughout life. This holds an invaluable potential to understand, and treat, a wide range of plasticity-related neurological disorders.Contents . . . vii List of Abbreviations . . . xv Summary . . . xxi Samenvatting . . . xxv List of Figures . . . xxix List of Tables . . . xxxiii Aim of the study . . . 1 1 General introduction . . . 5 1.1 An introduction to brain plasticity . . . 6 1.1.1 Experience-dependent plasticity in health and disease . . . 6 1.1.2 Multiple levels of brain plasticity . . . 10 1.2 The mouse visual system . . . 16 1.2.1 Extracting visual information from the world: from retina to cortex . . . 17 1.2.2 The mouse visual cortex . . . 19 1.3 Visual cortex plasticity . . . 29 1.3.1 Ocular dominance plasticity: the importance of inhibition . . . 30 1.3.2 Cross-modal plasticity as a response to sensory loss . . . 35 2 The current status of somatostatin-interneurons in inhibitory control of brain function and plasticity . . . 43 2.1 Introduction . . . 44 2.2 Interneurons in the mammalian neocortex . . . 46 2.3 Subdivision of SST-positive inhibitory neurons . . . 48 2.3.1 Martinotti cells . . . 48 2.3.2 Distinct SST-subpopulations: GIN-, X98-, and X94- mouse strains . . . 51 2.3.3 Distinct SST-subpopulations within the GIN-strain . . . 52 2.3.4 Long-distance projecting SST-inhibitory neurons . . . 54 2.3.5 Distinct functional SST-subtypes based on differences in ion channels and calcium-binding proteins . . . 54 2.4 Connecting the dots: SST-interneurons in the general cortical connectivity scheme and functional implications in cortical information processing . . . 55 2.4.1 Chemical synapses . . . 55 2.4.2 The input/output relationship of distinct SST-subpopulations . . . 56 2.4.3 Electrical coupling . . . 61 2.4.4 Cholinergic modulation of SST-interneurons . . . 61 2.5 Implications of SST-interneurons in experience-dependent cortical plasticity . . . 62 2.5.1 Implications of SST-interneurons in learning-induced plasticity . . . 63 2.5.2 Plasticity in the sensory deprived brain: SST-interneurons in ocular dominance plasticity . . . 65 2.5.3 SST-interneurons as potential integrators during crossmodal plasticity . . . 68 2.6 Outlook . . . 73 3 Evaluation of the expression pattern of rAAV2/1, 2/5, 2/7, 2/8, and 2/9 serotypes with different promoters in the mouse visual cortex . . . 75 3.1 Introduction . . . 76 3.2 Materials and methods . . . 78 3.2.1 Viral vector production . . . 78 3.2.2 Vector injections . . . 79 3.2.3 Tissue preparation and histology . . . 80 3.2.4 Antibody characterization . . .80 3.2.5 Visualization of expression patterns with DAB staining . . . 82 3.2.6 Immunohistochemistry . . . 83 3.2.7 Nissl staining . . . 85 3.2.8 Cell counting: laminar distribution of expression . . . 86 3.2.9 Cell counting: cell type-specific expression . . . 86 3.2.10 Statistical analysis . . . 87 3.3 Results . . . 88 3.3.1 Specific rAAV2 pseudotypes result in unique transduction patterns in the visual cortex . . . 88 3.3.2 Specific rAAV2 pseudotypes describe a unique laminar distribution . . . 92 3.3.3 Observations of anterograde and retrograde transport of rAAV2 pseudotypes . . . 92 3.3.4 Evaluation of promoter-specific expression in excitatory and inhibitory neuronal populations . . . 97 3.3.5 CaMKIIa1.3 promoter is less specific for excitatory neurons than CaMKIIa0.4 . . . 101 3.4 Discussion . . . 107 3.4.1 Viral vector expression pattern . . . 107 3.4.2 Laminar distribution of expression . . . 109 3.4.3 Anterograde and retrograde transport . . . 110 3.4.4 Cell type-specific transduction . . . 111 3.4.5 Conclusions . . . 112 4 Transient and localized optogenetic activation of somatostatininterneurons in mouse visual cortex abolishes long-term cortical plasticity due to vision loss . . . 113 4.1 Introduction . . . 114 4.2 Materials and methods . . . 117 4.2.1 Animals . . . 117 4.2.2 Viral vectors . . . 117 4.2.3 Verifying the specificity and expression pattern of the viral vector transduction . . . 118 4.2.4 Patch clamp whole-cell recordings to validate the functionality of the SSFOs . . . 119 4.2.5 Chronic implantation for light-delivery . . . 120 4.2.6 SSFO-stimulation protocol in freely moving animals . . . 121 4.2.7 Visual deprivation paradigm and tissue preparation . . . 122 4.2.8 In situ hybridization for zif268-mRNA . . . 123 4.2.9 Histology and localization of visual areal boundaries with Nissl patterns . . . 124 4.2.10 Quantitative analysis of ISH results . . . 125 4.2.11 Statistics . . . 127 4.3 Results . . . 127 4.3.1 Validation of cell-type specific SSFO expression, and functionality in SST-interneurons in adult mouse visual cortex . . . 129 4.3.2 Zif268 expression in visual cortex is not affected by optic fiber implantation . . . 130 4.3.3 SST-interneuron stimulation, DE, and the combination of both pretreatment strategies prior to ME induction impede the reactivation of the visual cortex . . . 132 4.3.4 SST-interneuron stimulation does not affect the response of non-visual cortical areas to long-term ME, in contrast to DE-pretreatment . . . 140 4.4 Discussion . . . 142 4.4.1 SST-interneuron stimulation prevents cross-modal takeover of visually deprived cortex . . . 142 4.4.2 Combining SST-interneuron stimulation and DE results in a more severe loss of cortical reactivation . . . 144 4.4.3 Possible mechanism of SST-interneuron mediated inhibitory regulation of plasticity . . . 145 4.4.4 Circuit level of SST-interneuron modulation of plasticity . . . 146 4.4.5 Conclusion . . . 148 4.5 Appendix . . . 149 5 Silencing somatostatin- interneuron activity during cross-modal reorganization: a chemogenetic approach . . . 151 5.1 Introduction . . . 152 5.2 Materials and methods . . . 155 5.2.1 Animals . . . 155 5.2.2 Viral vectors . . . 156 5.2.3 Activation of hM3Dq and hM4Di DREADDs in excitatory neurons . . . 157 5.2.4 Visual deprivation paradigm and chronic silencing of SSTinterneurons in awake and freely moving animals . . . 158 5.2.5 Tissue preparation . . . 158 5.2.6 Quantitative analysis of in situ hybridization for zif268 - mRNA . . . 159 5.2.7 Immunohistochemistry . . . 161 5.2.8 Statistics . . . 162 5.3 Results . . . 163 5.3.1 Optimal CNO-concentration to reliably activate or silence excitatory neurons following hM3Dq- or hM4Di-activation . . . 163 5.3.2 Silencing SST-interneurons around ME-onset results in increased plastic recovery following long-term ME . . . 165 5.3.3 Silencing SST-interneurons during the cross-modal recovery window following ME does not influence plastic reorganization but overrides DE-pretreatment effects . . . 168 5.3.4 hM4Di expression induces reactive astrogliosis . . . 169 5.4 Discussion . . . 173 5.4.1 Summary . . . 173 5.4.2 SST-interneuron silencing combined with ME results in strong reactivation in the deprived visual cortex . . . 173 5.4.3 Silencing SST-interneurons after sensory loss has occurred still boosts the cortical reactivation potential . . . 175 5.4.4 Possible mechanism for V1 SST-interneurons to control cross-modal plasticity . . . 177 5.4.5 hM4Di expression results in astrogliosis at the injection site . . . 178 5.4.6 Outlook . . . 179 6 General discussion and future perspectives . . . 181 6.1 Concluding remarks on the effects of SST-interneuron manipulation on adult cross-modal plasticity . . . 181 6.2 Possible mechanisms of SST-interneuron mediated control over cross-modal plasticity . . . 184 6.2.1 Where do SST-interneurons receive their inputs from? . . . 184 6.2.2 SST-interneurons in V1 versus the extrastriate regions: gateways for the invasion of cross-modal inputs? . . . 188 6.2.3 Molecular changes in SST-interneurons . . . 189 6.3 Future perspectives: where to go from here with SST-interneurons . . . 190 6.3.1 Some considerations on the use of DREADDs . . . 190 6.3.2 Functional assessment of visual cortex recovery to distinguish open-eye potentiation from cross-modal recovery . . . 192 6.3.3 Upstream targets: VIP-interneurons and cholinergic neuromodulation . . . 193 6.3.4 Next-generation single-cell RNA-sequencing approaches to overcome neuronal heterogeneity . . . 194 6.4 Clinical relevance for an on/off switch for cortical plasticity . . . 195 Bibliography . . . 199 Curriculum vitae . . . 251 List of Publications . . . 253nrpages: 296status: publishe

The current status of somatostatin-interneurons in inhibitory control of brain function and plasticity

The mammalian neocortex contains many distinct inhibitory neuronal populations to balance excitatory neurotransmission. A correct excitation/inhibition equilibrium is crucial for normal brain development, functioning, and controlling lifelong cortical plasticity. Knowledge about how the inhibitory network contributes to brain plasticity however remains incomplete. Somatostatin- (SST-) interneurons constitute a large neocortical subpopulation of interneurons, next to parvalbumin- (PV-) and vasoactive intestinal peptide- (VIP-) interneurons. Unlike the extensively studied PV-interneurons, acknowledged as key components in guiding ocular dominance plasticity, the contribution of SST-interneurons is less understood. Nevertheless, SST-interneurons are ideally situated within cortical networks to integrate unimodal or cross-modal sensory information processing and therefore likely to be important mediators of experience-dependent plasticity. The lack of knowledge on SST-interneurons partially relates to the wide variety of distinct subpopulations present in the sensory neocortex. This review informs on those SST-subpopulations hitherto described based on anatomical, molecular, or electrophysiological characteristics and whose functional roles can be attributed based on specific cortical wiring patterns. A possible role for these subpopulations in experience-dependent plasticity will be discussed, emphasizing on learning-induced plasticity and on unimodal and cross-modal plasticity upon sensory loss. This knowledge will ultimately contribute to guide brain plasticity into well-defined directions to restore sensory function and promote lifelong learning.status: publishe

Visual system plasticity in mammals: the story of enucleation-induced vision loss

The groundbreaking work of Hubel and Wiesel in the 1960's on ocular dominance plasticity instigated many studies of the visual system of mammals, enriching our understanding of how the development of its structure and function depends on high quality visual input through both eyes. These studies have mainly employed lid suturing, dark rearing and eye patching applied to different species to reduce or impair visual input, and have created extensive knowledge on binocular vision. However, not all aspects and types of plasticity in the visual cortex have been covered in full detail. In that regard, a more drastic deprivation method like enucleation, leading to complete vision loss appears useful as it has more widespread effects on the afferent visual pathway and even on non-visual brain regions. One-eyed vision due to monocular enucleation (ME) profoundly affects the contralateral retinorecipient subcortical and cortical structures thereby creating a powerful means to investigate cortical plasticity phenomena in which binocular competition has no vote.In this review, we will present current knowledge about the specific application of ME as an experimental tool to study visual and cross-modal brain plasticity and compare early postnatal stages up into adulthood. The structural and physiological consequences of this type of extensive sensory loss as documented and studied in several animal species and human patients will be discussed. We will summarize how ME studies have been instrumental to our current understanding of the differentiation of sensory systems and how the structure and function of cortical circuits in mammals are shaped in response to such an extensive alteration in experience. In conclusion, we will highlight future perspectives and the clinical relevance of adding ME to the list of more longstanding deprivation models in visual system research.status: publishe

A tool for brain-wide quantitative analysis of molecular data upon projection into a planar view of choice

Several techniques, allowing the reconstruction and visualization of functional, anatomical or molecular information from tissue and organ slices, have been developed over the years. Yet none allow direct comparison without reprocessing the same slices. Alternative methods using publicly available reference maps like the Allen Brain Atlas lack flexibility with respect to age and species. We propose a new approach to reconstruct a segmented region of interest from serial slices by projecting the optical density values representing a given molecular signal to a plane of view of choice, and to generalize the results into a reference map, which is built from the individual maps of all animals under study. Furthermore, to allow quantitative comparison between experimental conditions, a non-parametric pseudo t-test has been implemented. This new mapping tool was applied, optimized and validated making use of an in situ hybridization dataset that represents the spatiotemporal expression changes for the neuronal activity reporter gene zif268, in relation to cortical plasticity induced by monocular enucleation, covering the entire mouse visual cortex. The created top view maps of the mouse brain allow precisely delineating and interpreting 11 extrastriate areas surrounding mouse V1. As such, and because of the opportunity to create a planar projection of choice, these molecular maps can in the future easily be compared with functional or physiological imaging maps created with other techniques such as Ca2+, flavoprotein and optical imaging.status: publishe

c-Fos expression following context conditioning and deep brain stimulation in the bed nucleus of the stria terminalis in rats

Deep brain stimulation (DBS) in the bed nucleus of the stria terminalis (BST), a region implicated in the expression of anxiety, shows promise in psychiatric patients, but its effects throughout the limbic system are largely unknown. In male Wistar rats, we first evaluated the neural signature of contextual fear (N = 16) and next, of the anxiolytic effects of high-frequency electrical stimulation in the BST (N = 31), by means of c-Fos protein expression. In non-operated animals, we found that the left medial anterior BST displayed increased c-Fos expression in anxious (i.e., context-conditioned) versus control subjects. Moreover, control rats showed asymmetric expression in the basolateral amygdala (BLA) (i.e., higher intensities in the right hemisphere), which was absent in anxious animals. The predominant finding in rats receiving bilateral BST stimulation was a striking increase in c-Fos expression throughout much of the left hemisphere, which was not confined to the predefined regions of interest. To conclude, we found evidence for lateralized c-Fos expression during the expression of contextual fear and anxiolytic high-frequency electrical stimulation of the BST, particularly in the medial anterior BST and BLA. In addition, we observed an extensive and unexpected left-sided c-Fos spread following bilateral stimulation in the BST.status: publishe