Cholinergic modulation of striatal microcircuits.

The purpose of this review is to bridge the gap between earlier literature on striatal cholinergic interneurons and mechanisms of microcircuit interaction demonstrated with the use of newly available tools. It is well known that the main source of the high level of acetylcholine in the striatum, compared to other brain regions, is the cholinergic interneurons. These interneurons provide an extensive local innervation that suggests they may be a key modulator of striatal microcircuits. Supporting this idea requires the consideration of functional properties of these interneurons, their influence on medium spiny neurons, other interneurons, and interactions with other synaptic regulators. Here, we underline the effects of intrastriatal and extrastriatal afferents onto cholinergic interneurons and discuss the activation of pre- and postsynaptic muscarinic and nicotinic receptors that participate in the modulation of intrastriatal neuronal interactions. We further address recent findings about corelease of other transmitters in cholinergic interneurons and actions of these interneurons in striosome and matrix compartments. In addition, we summarize recent evidence on acetylcholine-mediated striatal synaptic plasticity and propose roles for cholinergic interneurons in normal striatal physiology. A short examination of their role in neurological disorders such as Parkinson's, Huntington's, and Tourette's pathologies and dystonia is also included.

Thalamic afferents to prefrontal cortices from ventral motor nuclei in decision-making.

The focus of this literature review is on the three interacting brain areas that participate in decision-making: basal ganglia, ventral motor thalamic nuclei, and medial prefrontal cortex, with an emphasis on the participation of the ventromedial and ventral anterior motor thalamic nuclei in prefrontal cortical function. Apart from a defining input from the mediodorsal thalamus, the prefrontal cortex receives inputs from ventral motor thalamic nuclei that combine to mediate typical prefrontal functions such as associative learning, action selection, and decision-making. Motor, somatosensory and medial prefrontal cortices are mainly contacted in layer 1 by the ventral motor thalamic nuclei and in layer 3 by thalamocortical input from mediodorsal thalamus. We will review anatomical, electrophysiological, and behavioral evidence for the proposed participation of ventral motor thalamic nuclei and medial prefrontal cortex in rat and mouse motor decision-making.

An Introspective Approach: A Lifetime of Parkinson's Disease Research and Not Much to Show for it Yet?

I feel part of a massive effort to understand what is wrong with motor systems in the brain relating to Parkinson's disease. Today, the symptoms of the disease can be modified slightly, but dopamine neurons still die; the disease progression continues inexorably. Maybe the next research phase will bring the power of modern genetics to bear on halting, or better, preventing cell death. The arrival of accessible human neuron assemblies in organoids perhaps will provide a better access to the processes underlying neuronal demise.

In Vivo Wireless Optogenetic Control of Skilled Motor Behavior.

Fine motor skills are essential in everyday life and can be compromised in several nervous system disorders. The acquisition and performance of these tasks require sensory-motor integration and involve precise control of bilateral brain circuits. Implementing unimanual behavioral paradigms in animal models will improve the understanding of the contribution of brain structures, like the striatum, to complex motor behavior as it allows manipulation and recording of neural activity of specific nuclei in control conditions and disease during the performance of the task. Since its creation, optogenetics has been a dominant tool for interrogating the brain by enabling selective and targeted activation or inhibition of neuronal populations. The combination of optogenetics with behavioral assays sheds light on the underlying mechanisms of specific brain functions. Wireless head-mounted systems with miniaturized light-emitting diodes (LEDs) allow remote optogenetic control in an entirely free-moving animal. This avoids the limitations of a wired system being less restrictive for animals' behavior without compromising light emission efficiency. The current protocol combines a wireless optogenetics approach with high-speed videography in a unimanual dexterity task to dissect the contribution of specific neuronal populations to fine motor behavior.

Striatal bilateral control of skilled forelimb movement.

Skilled motor behavior requires bihemispheric coordination, and participation of striatal outputs originating from two neuronal groups identified by distinctive expression of D1 or D2 dopamine receptors. We trained mice to reach for and grasp a single food pellet and determined how the output pathways differently affected forelimb trajectory and task efficiency. We found that inhibition and excitation of D1-expressing spiny projection neurons (D1SPNs) have a similar effect on kinematics results, as if excitation and inhibition disrupt the whole ensemble dynamics and not exclusively one kind of output. In contrast, D2SPNs participate in control of target accuracy. Further, ex vivo electrophysiological comparison of naive mice and mice exposed to the task showed stronger striatal neuronal connectivity for ipsilateral D1 and contralateral D2 neurons in relation to the paw used. In summary, while the output pathways work together to smoothly execute skill movements, practice of the movement itself changes synaptic patterns.

Striatal interneurons in dissociated cell culture.

In addition to the well-characterized direct and indirect projection neurons there are four major interneuron types in the striatum. Three contain GABA and either parvalbumin, calretinin or NOS/NPY/somatostatin. The fourth is cholinergic. It might be assumed that dissociated cell cultures of striatum (typically from embryonic day E18.5 in rat and E14.5 for mouse) contain each of these neuronal types. However, in dissociated rat striatal (caudate/putamen, CPu) cultures arguably the most important interneuron, the giant aspiny cholinergic neuron, is not present. When dissociated striatal neurons from E14.5 Sprague-Dawley rats were mixed with those from E18.5 rats, combined cultures from these two gestational periods yielded surviving cholinergic interneurons and representative populations of the other interneuron types at 5 weeks in vitro. Neurons from E12.5 CD-1 mice were combined with CPu neurons from E14.5 mice and the characteristics of striatal interneurons after 5 weeks in vitro were determined. All four major classes of interneurons were identified in these cultures as well as rare tyrosine hydroxylase positive interneurons. However, E14.5 mouse CPu cultures contained relatively few cholinergic interneurons rather than the nearly total absence seen in the rat. A later dissection day (E16.5) was required to obtain mouse CPu cultures totally lacking the cholinergic interneuron. We show that these cultures generated from two gestational age cells have much more nearly normal proportions of interneurons than the more common organotypic cultures of striatum. Interneurons are generated from both ages of embryos except for the cholinergic interneurons that originate from the medial ganglionic eminence of younger embryos. Study of these cultures should more accurately reflect neuronal processing as it occurs in the striatum in vivo. Furthermore, these results reveal a procedure for parallel culture of striatum and cholinergic depleted striatum that can be used to examine the function of the cholinergic interneuron in striatal networks.

Selective elimination of glutamatergic synapses on striatopallidal neurons in Parkinson disease models.

Parkinson disease is a common neurodegenerative disorder that leads to difficulty in effectively translating thought into action. Although it is known that dopaminergic neurons that innervate the striatum die in Parkinson disease, it is not clear how this loss leads to symptoms. Recent work has implicated striatopallidal medium spiny neurons (MSNs) in this process, but how and precisely why these neurons change is not clear. Using multiphoton imaging, we show that dopamine depletion leads to a rapid and profound loss of spines and glutamatergic synapses on striatopallidal MSNs but not on neighboring striatonigral MSNs. This loss of connectivity is triggered by a new mechanism-dysregulation of intraspine Cav1.3 L-type Ca(2+) channels. The disconnection of striatopallidal neurons from motor command structures is likely to be a key step in the emergence of pathological activity that is responsible for symptoms in Parkinson disease.

Prelimbic cortical targets of ventromedial thalamic projections include inhibitory interneurons and corticostriatal pyramidal neurons in the rat.

Ventromedial thalamic axons innervate cortical layer I and make contacts onto the apical dendritic tuft of pyramidal neurons. Optical stimulation of ventromedial thalamic axon terminals in prefrontal cortical areas in mouse brain slices evokes responses in corticocortical, corticothalamic and layer I inhibitory interneurons. Using anterograde tracing techniques and immunohistochemistry in male Sprague-Dawley rats, we provide anatomical evidence that ventromedial thalamic axon terminals in prelimbic cortex make contacts onto pyramidal neurons and, in particular, onto corticostriatal neurons as well as layer I inhibitory interneurons. Using stereology, we made quantitative estimates of contacts in uppermost prelimbic layer I onto dendrites of pyramidal neurons, corticostriatal neurons and layer I inhibitory interneurons. Prefrontal cortex has long been associated with decision making. Specifically, corticostriatal neurons in rat prelimbic cortex play an important role in cost-benefit decision making. Although recent experiments have detailed the physiology of this area in thalamocortical circuits, the extent of the impact of ventromedial thalamic input on corticostriatal neurons or layer I inhibitory interneurons has not been explored. Our quantitative anatomical results provide evidence that most ventromedial thalamic input to pyramidal neurons is provided to corticostriatal neurons and that overall more contacts are made onto the population of excitatory than onto the population of inhibitory neurons.

Space, time and dopamine.

In recent years, dopamine has emerged as a key neurotransmitter that is crucially involved in incentive motivation and reinforcement learning. Dopamine release is evoked by rewards. The extensive divergence of outputs from a small number of dopaminergic neurons suggests a spatially nonselective action of dopamine, but it reinforces the specific actions that led to reward. How is this achieved? We propose that the selectivity of dopamine effects is achieved by the timing of dopamine release in relation to the activity of glutamatergic synapses, rather than by spatial localization of the dopamine signal to specific synaptic contacts. The synaptic mechanisms of these actions are unknown but reduced levels of dopamine, for example in Parkinson's disease, leads to a paucity of behavioural output, whereas its excess production has been associated with psychiatric problems. Clearly, there are therapeutic imperatives that require a better understanding of how dopamine functions at a synaptic level.

Slowly progressive dopamine cell loss--a model on which to test neuroprotective strategies for Parkinson's disease?

Making animal models of human disease is a very flawed process. Aspects of the disease can be imitated but models do not necessarily give reliable leads for treatment strategies. When Ungerstedt in Sweden first described the 6-hydroxydopamine (6-OHDA) treated rat model of Parkinson's disease /89/ we knew that the symptoms would not map readily to those of the human disease--rats have four legs after all. On the other hand, the neuropathology looked exactly like end-stage Parkinsonian pathology. That remained true even as we explored other types of neuropathology in the rats /24,43-46,80/. Many of today's treatments for Parkinsonism are developed from pharmacological studies on that model of rats with a chemically induced lesion. However, the 6-OHDA model does not address the important issue of a cure for the disease. The triggers and the time-course of dopamine (DA) cell death in rats are known for nearly every disease model - but for the human disease there is no equivalent knowledge. In the human, the neurons have been dying for a considerable time before the symptoms become obvious and they go on dying even with adequate symptomatic relief /94/, but after intracerebral administration of 6-OHDA to an animal the cells die quickly; all cells are destroyed in less than 5 days /42,88,89/. Thus, we were interested in developing an animal model of DA cell death with a slower time-course. After ibotenic acid injections into rat globus pallidus (GP), DA cells are lost from the ipsilateral substantia nigra over the slower time scale of about six weeks. This time scale has allowed us to test some interventions to prevent the cells from dying. Although some attempts have succeeded, cell death is prevented only for three weeks -beyond that treatments fail and DA cells die. At the moment, this model has at least opened a window into causes of neuronal death in a slower time scale /94/ than previous rodent models.

Simulation of GABA function in the basal ganglia: computational models of GABAergic mechanisms in basal ganglia function.

This chapter outlines current interpretation of computational aspects of GABAergic circuits of the striatum. Recent hypotheses and controversial matters are reviewed. Quantitative aspects of striatal synaptology relevant to computational models are considered, with estimates of the connectivity of the spiny projection neurons and fast-spiking interneurons. Against this background, insights into the computational properties of inhibitory circuits based on analysis and simulation of simple models are discussed. The paper concludes with suggestions for further theoretical and experimental studies.

Striatal contributions to reward and decision making: making sense of regional variations in a reiterated processing matrix.

The striatum is the major input nucleus of the basal ganglia. It is thought to play a key role in learning on the basis of positive reinforcement and in action selection. One view of the striatum conceives it as comprising a reiterated matrix of processing units that perform common operations in different striatal regions, namely synaptic plasticity according to a three-factor rule, and lateral inhibition. These operations are required for reinforcement learning and selection of previously reinforced actions. Analysis of the behavioral effects of circumscribed lesions of the striatum, however, suggests regional specialization of learning and decision-making operations. We consider how a basic processing unit may be modified by regional variations in neurochemical parameters, for example, by the gradient in density of dopamine terminals from dorsal to ventral striatum. These variations suggest subtle differences between dorsolateral and ventromedial striatal regions in the temporal properties of dopamine signaling, which are superimposed on regional differences in connectivity. We propose that these variations make sense in relation to the temporal structure of activity in striatal inputs from different regions, and the requirements of different learning operations. Dorsolateral striatal (DLS) regions may be subject to brief, precisely timed pulses of dopamine, whereas ventromedial striatal regions integrate dopamine signals over a longer time course. These differences may be important for understanding regional variations in the contribution to reinforcement of habits, versus incentive processes that are sensitive to the value of expected rewards.

Delayed synaptic degeneration in the CNS of Wlds mice after cortical lesion.

Therapies that might delay degeneration of synapses offer an appealing strategy for treatment of neurodegenerative diseases, including Alzheimer's disease and related dementias, prion diseases, schizophrenia and amyotrophic lateral sclerosis. Analysis of mouse mutants provides one possible avenue towards identifying relevant mechanisms. Here, we used quantitative and serial section electron microscopy to find out whether the onset and time course of pre-synaptic nerve terminal degeneration is delayed in the striatum of Wallerian degeneration slow (Wld(s)) mutant mice. Synaptic degeneration was observed within 48 h of cortical ablation in wild-type mice but was delayed by approximately 1 week in Wld(s) mice. However, the morphological characteristics of degenerating nerve terminals in wild-type and Wld(s) mice were indistinguishable, in contrast to the differences reported previously in studies of the PNS. Surprisingly, the delayed onset of synaptic degeneration was accompanied by an increased incidence of complex synaptic morphologies on post-synaptic spines in the denervated Wld(S) striatum indicating an enhanced plastic response at both injured and uninjured synapses. The data suggest that targeting Wallerian-like mechanisms of synaptic degeneration could lead to the development of new therapies for the treatment of CNS disorders where synapse loss is a primary feature.

Are the Symptoms of Parkinsonism Cortical in Origin?

We present three reasons to suspect that the major deleterious consequence of dopamine loss from the striatum is a cortical malfunction. We suggest that it is cortex, rather than striatum, that should be considered as the source of the debilitating symptoms of Parkinson's disease (PD) since:1.Cortical synapses onto striatal dendritic spines are lost in PD.2.All known treatments of the symptoms of PD disrupt beta oscillations. Oscillations that are also disrupted following antidromic activation of cortical neurons.3.The final output of basal ganglia directly modulates thalamic connections to layer I of frontal cortical areas, regions intimately associated with motor behaviour. These three reasons combined with evidence that the current summary diagram of the basal ganglia involvement in PD is imprecise at best, suggest that a re-orientation of the treatment strategies towards cortical, rather than striatal malfunction, is overdue.

Neurone specific regulation of dendritic spines in vivo by post synaptic density 95 protein (PSD-95).

Post synaptic density protein 95 (PSD-95) is a postsynaptic adaptor protein coupling the NMDA receptor to downstream signalling pathways underlying plasticity. Mice carrying a targeted gene mutation of PSD-95 show altered behavioural plasticity including spatial learning, neuropathic pain, orientation preference in visual cortical cells, and cocaine sensitisation. These behavioural effects are accompanied by changes in long-term potentiation of synaptic transmission. In vitro studies of PSD-95 signalling indicate that it may play a role in regulating dendritic spine structure. Here, we show that PSD-95 mutant mice have alterations in dendritic spine density in the striatum (a 15% decrease along the dendritic length) and in the hippocampus (a localised 40% increase) without changes in dendritic branch patterns or gross neuronal architecture. These changes in spine density were accompanied by altered expression of proteins known to interact with PSD-95, including NR2B and SAP102, suggesting that PSD-95 plays a role in regulating the expression and activation of proteins found within the NMDA receptor complex. Thus, PSD-95 is an important regulator of neuronal structure as well as plasticity in vivo.

Basal ganglia-thalamus and the "crowning enigma".

When Hubel (1982) referred to layer 1 of primary visual cortex as "… a 'crowning mystery' to keep area-17 physiologists busy for years to come …" he could have been talking about any cortical area. In the 80's and 90's there were no methods to examine this neuropile on the surface of the cortex: a tangled web of axons and dendrites from a variety of different places with unknown specificities and doubtful connections to the cortical output neurons some hundreds of microns below. Recently, three changes have made the crowning enigma less of an impossible mission: the clear presence of neurons in layer 1 (L1), the active conduction of voltage along apical dendrites and optogenetic methods that might allow us to look at one source of input at a time. For all of those reasons alone, it seems it is time to take seriously the function of L1. The functional properties of this layer will need to wait for more experiments but already L1 cells are GAD67 positive, i.e., inhibitory! They could reverse the sign of the thalamic glutamate (GLU) input for the entire cortex. It is at least possible that in the near future normal activity of individual sources of L1 could be detected using genetic tools. We are at the outset of important times in the exploration of thalamic functions and perhaps the solution to the crowning enigma is within sight. Our review looks forward to that solution from the solid basis of the anatomy of the basal ganglia output to motor thalamus. We will focus on L1, its afferents, intrinsic neurons and its influence on responses of pyramidal neurons in layers 2/3 and 5. Since L1 is present in the whole cortex we will provide a general overview considering evidence mainly from the somatosensory (S1) cortex before focusing on motor cortex.

Rebuilding a realistic corticostriatal "social network" from dissociated cells.

Many of the methods available for the study of cortical influences on striatal neurons have serious problems. In vivo the connectivity is so complex that the study of input from an individual cortical neuron to a single striatal cell is nearly impossible. Mixed corticostriatal cultures develop many connections from striatal cells to cortical cells, in striking contrast to the fact that only connections from cortical cells to striatal cells are present in vivo. Furthermore, interneuron populations are over-represented in organotypic cultures. For these reasons, we have developed a method for growing cortical and striatal neurons in separated compartments that allows cortical neurons to innervate striatal cells in culture. The method works equally well for acutely dissociated or cryopreserved neurons and allows a number of manipulations that are not otherwise possible. Either cortical or striatal compartments can be transfected with channel rhodopsins. The activity of both areas can be recorded in multielectrode arrays or individual patch recordings from pairs of cells. Finally, corticostriatal connections can be severed acutely. This procedure enables determination of the importance of corticostriatal interaction in the resting pattern of activity. These cultures also facilitate development of sensitive analytical network methods to track connectivity.

Cell Assembly Signatures Defined by Short-Term Synaptic Plasticity in Cortical Networks.

The cell assembly (CA) hypothesis has been used as a conceptual framework to explain how groups of neurons form memories. CAs are defined as neuronal pools with synchronous, recurrent and sequential activity patterns. However, neuronal interactions and synaptic properties that define CAs signatures have been difficult to examine because identities and locations of assembly members are usually unknown. In order to study synaptic properties that define CAs, we used optical and electrophysiological approaches to record activity of identified neurons in mouse cortical cultures. Population analysis and graph theory techniques allowed us to find sequential patterns that represent repetitive transitions between network states. Whole cell pair recordings of neurons participating in repeated sequences demonstrated that synchrony is exhibited by groups of neurons with strong synaptic connectivity (concomitant firing) showing short-term synaptic depression (STD), whereas alternation (sequential firing) is seen in groups of neurons with weaker synaptic connections showing short-term synaptic facilitation (STF). Decreasing synaptic weights of a network promoted the generation of sequential activity patterns, whereas increasing synaptic weights restricted state transitions. Thus in simple cortical networks of real neurons, basic signatures of CAs, the properties that underlie perception and memory in Hebb's original description, are already present.

Extrasynaptic glutamate NMDA receptors: key players in striatal function.

N-methyl-D-aspartate receptors (NMDAR) are crucial for the function of excitatory neurotransmission and are present at the synapse and on the extrasynaptic membrane. The major nucleus of the basal ganglia, striatum, receives a large glutamatergic excitatory input carrying information about movements and associated sensory stimulation for its proper function. Such bombardment of glutamate synaptic release results in a large extracellular concentration of glutamate that can overcome the neuronal and glial uptake homeostatic systems therefore allowing the stimulation of extrasynaptic glutamate receptors. Here we have studied the participation of their extrasynaptic type in cortically evoked responses or in the presence of NMDARs stimulation. We report that extrasynaptic NMDAR blocker memantine, reduced in a dose-dependent manner cortically induced NMDA excitatory currents in striatal neurons (recorded in zero-Mg(++) plus DNQX 10 µM). Moreover, memantine (2-4 µM) significantly reduced the NMDAR-dependent membrane potential oscillations called up and down states. Recordings of neuronal striatal networks with a fluorescent calcium indicator or with multielectrode arrays (MEA) also showed that memantine reduced in a dose-dependent manner, NMDA-induced excitatory currents and network behavior. We used multielectrode arrays (MEA) to grow segregated cortical and striatal neurons. Once synaptic contacts were developed (>21DIV) recordings of extracellular activity confirmed the cortical drive of spontaneous synchronous discharges in both compartments. After severing connections between compartments, active striatal neurons in the presence of memantine (1 µM) and CNQX (10 µM) were predominantly fast spiking interneurons (FSI). The significance of extrasynaptic receptors in the regulation of striatal function and neuronal network activity is evident.