Cholinergic Interneurons: Distribution and Synaptic Inputs in the Primate Putamen Open Access

Gonzales, Kalynda Kari (2013)

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Striatal cholinergic interneurons (ChIs) are central for the processing and reinforcement of goal-directed and habitual reward-related behaviors that can be negatively affected in states of altered dopamine transmission such as in Parkinson's disease. Although significant advances have been made in understanding the mechanisms by ChIs involvement in such behaviors, the development of potential therapeutic interventions that target ChI activity has been hampered by our limited knowledge about the network of connections that target ChIs. Anatomical, pharmacological, and electrophysiological studies in rodents and primates have demonstrated that striatal ChI activity is modulated by GABAergic and glutamatergic inputs. However, the source(s) and prevalence of these inputs have not been clarified. The main objective of this thesis was therefore to perform a quantitative ultrastructural analysis of the GABAergic inputs from direct (substance P-containing) and indirect (enkephalin-containing) striatofugal neurons, as well as inputs from GABAergic and glutamatergic parvalbumin-containing neurons, onto ChIs in the monkey putamen.

Electron microscopic observations from double-labeled (i.e., immunogold and immunoperoxidase) tissue revealed that approximately 60% of all synaptic inputs to ChIs originate from GABAergic terminals, where 24% of this innervation is derived from axon collaterals of direct and indirect striatal projection neurons, and 10% from striatal and/or pallidal GABAergic parvalbumin-containing neurons. Of the remaining synaptic inputs to ChIs, 21% are putatively glutamatergic, half of which originate from parvalbumin-containing neurons in the thalamus, and 19% from other (non-GABAergic) sources. The distribution of these synaptic inputs to striatal ChIs follows two major patterns: (1) GABAergic terminals that express substance P or enkephalin terminate on the entire somatodendritic domain of ChIs, whereas (2) medium- and small-sized dendrites of ChIs are targets of parvalbumin-containing GABAergic and glutamatergic inputs.

Altogether, these studies have revealed an extensively diverse synaptic network between ChIs and their GABAergic and glutamatergic afferents, resulting in the first detailed map of ChI connectivity in the monkey striatum. Future studies in relation to the prevalence and pattern of additional ChI afferents, combined with a detailed description of changes in this afferent synaptic network in pathological conditions, will help to further characterize the substrate that underlies the role of ChIs in striatal functions in normal and diseased states.

Table of Contents

Abstract iii Acknowledgements vi

Table of Contents vii

List of Figures xi

List of Tables xii

Chapter 1 : Introduction & Background 1

1.1 Introduction 2

1.2 Striatum: functional compartmentalization and afferent connections 4

1.2.1 Striatal compartmentalization 4

1.2.2 Corticostriatal system 5

1.2.3 Thalamostriatal system 6

1.2.4 Nigrostriatal and mesostriatal systems 6

1.2.5 Other striatal afferents 7

1.3 Striatum: cellular organization and efferent connections 7

1.3.1 Dorsal striatum 7

1.3.2 Striatal interneurons 8

1.4 Heterogeneity of striatal cholinergic interneurons 9

1.4.1 Cholinergic interneurons in the dorsal striatum 9 Morphological, ultrastructural, and cytological features 9 Dorsal striatal cholinergic neuropil 12

1.4.2 Physiological activity of cholinergic interneurons in the dorsal striatum 14

1.5 Cholinergic regulation of striatal activity 16

1.5.1 Cholinergic receptor expression in the dorsal striatum 16 Muscarinic receptor expression of striatal neurons 16 Nicotinic receptor expression of striatal neurons 19 Muscarinic and nicotinic receptor expression in striatal afferents 20 Electron microscopic localization of muscarinic and nicotinic receptors 21

1.5.2 Modulation of striatal activity by cholinergic receptors 23 Muscarinic and nicotinic regulation of cholinergic excitability in the striatum 23 Muscarinic and nicotinic modulation of GABAergic striatal neurons 24 Muscarinic modulation of striatal dopamine activity 27 Nicotinic modulation of striatal dopamine activity 28 Nicotinic modulation of striatal serotonin release 31

1.6 Synaptic regulation of striatal cholinergic interneurons 31

1.6.1 General synaptic innervation of cholinergic interneurons 31

1.6.2 Glutamatergic regulation of cholinergic interneurons 33 Glutamatergic receptors in cholinergic interneurons 34

1.6.3 GABAergic regulation of cholinergic interneurons 35

1.6.4 Peptidergic regulation of cholinergic interneurons 35

1.6.5 Dopaminergic regulation of cholinergic interneurons 36

1.7 Technical limitations of cholinergic receptor drugs, antibodies, and knock-out mice 38

1.8 Experimental design, rationale, and significance 39

Chapter 2 : GABAergic inputs from direct and indirect striatal projection neurons onto cholinergic interneurons in the primate putamen 40

2.1 Introduction 42

2.2 Materials and methods 43

2.2.1 Tissue processing 43

2.2.2 Primary antibodies 44

2.2.3 Single immunoperoxidase labeling for light microscopy 46

2.2.4 Preparation for electron microscopic observations 47 Single pre-embedding immunoperoxidase labeling for electron microscopy 47 Double post-embedding immunogold for GABA and pre-embedding immunoperoxidase for ChAT 48 Double pre-embedding immunoperoxidase for ChAT and pre-embedding immunogold for SP or Met-/Leu-Enk 49

2.2.5 Analysis of material 50 GABAergic inputs onto ChAT-positive interneurons 50 SP- or Met-/Leu-Enk-positive terminals onto ChAT-positive interneurons 51 Total synaptic innervation of ChAT-positive neurons from SP- or Enk-positive terminals 52

2.3 Results 53

2.3.1 Light microscopic observations 53 ChAT immunolabeling in the dorsal striatum 53 SP and Met-/Leu-Enk immunolabeling in the striatopallidal system 53

2.3.2 Electron microscopic observations 54 GABAergic innervation of ChAT-labeled neurons 55 SP-labeled inputs onto ChAT-immunoreactive neurons 59 Enk-positive terminals onto ChAT-positive neurons 61

2.4 Discussion 63

2.4.1 Synaptic GABAergic regulation of striatal cholinergic interneurons 63

2.4.2 Axon collaterals of striatal projection neurons are a main source of GABAergic inputs onto striatal cholinergic interneurons 66

2.4.3 Peptidergic modulation of cholinergic interneurons by striatal projection neurons? 67

Chapter 3 : Synaptic regulation of cholinergic interneurons: Inputs from parvalbumin-containing neurons in the primate putamen 69

3.1 Introduction 70

3.2 Materials and methods 72

3.2.1 Animals 72

3.2.2 Tissue Processing 72

3.2.3 Primary antibodies 73

3.2.4 Preparation for electron microscopic observations 74 Single pre-embedding immunoperoxidase labeling for electron microscopy 74 Double pre-embedding immunogold for PV and immunoperoxidase for ChAT 75

3.2.5 Analysis of material 75 PV-positive terminals targeting ChAT-positive interneurons 75 Total synaptic innervation of ChAT-positive neurons from PV-positive terminals 76 Dendritic densities in single- and double-labeled tissue 77

3.3 Results 79

3.3.1 General ultrastructural characterization of PV immunoreactivity in the striatum 79

3.3.2 Characterization of PV-labeled terminals in single and double immunoreactions 80 PV-positive terminals that form symmetric synapses 80 PV-labeled boutons that form asymmetric synapses 81 Total synaptic innervation of ChIs represented by PV-labeled terminals 83

3.4 Discussion 85

3.4.1 Symmetric versus asymmetric synapses onto ChIs 86

3.4.2 Intrastriatal and extrinsic PV-containing afferents of ChIs 87 PV-positive terminals forming symmetric synapses 88 PV-positive terminals forming asymmetric synapses 89

3.4.3 PV modulation of cholinergic interneurons by GABAergic and glutamatergic projections 90

Chapter 4 : Conclusions & Implications 93

4.1 Summary of main findings 94

4.2 Implications 95

4.2.1 New views on striatal circuitry of ChIs 95 GABA, neuropeptides, and the striatal cholinergic systems 95 ChIs in synaptic plasticity: roles of PV, calcium, and glia 97 Functional impact of the morphology and synaptic afferentation of ChI dendrites 98

4.3 Cholinergic interneuron dysfunction 100

4.3.1 Structural changes in ChI innervation in Parkinson's disease 100

4.3.2 Cholinergic therapies for Parkinson's disease 102

4.5 Concluding remarks 104

References 106

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