in-vivo-wireless-optogenetic-control-of-skilled-motor-behavior

<em>In Vivo</em> 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 ...

Okinawa Institute of Science and Technology Graduate University

Any simplified description of a brain system that survives 20 years is bound to have some problems. Twenty years of research should have added details that were not known when the now classical description of the basal ganglia circuitry was new. Some examples of such new information about the parts of the original model include: All of these details make it difficult to cling to the model as it was presented, but which of the new information will support a new synthesis? Do we have a coherent replacement for those powerfully heuristic ideas put forward by Albin Young and Penny in 1989? Perhaps in the quantitative anatomical detail there is a plan that may support a new theory. If all of the cortex is not accessible to every striatal cell. If dopamine influences many spines rather than the one it contacts. If we stop looking for a mechanical control system for movement in the basal ganglia and begin to investigate the predictive properties of 'reinforcement learning'. Then maybe a new functional model will emerge that is less tied to anatomical and physiological simplification, but perhaps will still allow us to engineer therapeutic strategies to help patients with movement disorders.

Dealing with the devil in the detail - some thoughts about the next model of the basal ganglia.

FRETing over dopamine: single cell cAMP and protein kinase A responses to 100 ms dopamine application.

High-frequency electrical stimulation that targets the subthalamic nucleus has proved to be beneficial in alleviating the motor symptoms in many patients with Parkinson disease. The mechanism of action for this paradigm of deep brain stimulation is still not fully understood, and this is, in part, attributed to the fact that there are diverse cellular elements at the stimulation site that could bring about local and distal effects. Recent studies in both human and animal models strongly suggest that the activity in the cortex, especially in the motor cortical areas, is directly altered by deep brain stimulation by signals traveling in an antidromic fashion from the subthalamic nucleus. Herein, we discuss the evidence for this proposition, as well as the mechanism by which antidromic activation desynchronizes motor cortical activity. The implications of these new findings for the pathogenesis and treatment of Parkinson disease are highlighted. 

Cortical effects of deep brain stimulation: implications for pathogenesis and treatment of Parkinson disease.

Over the years since the discovery of dopamine in the neostriatum, we have learned much about the anatomy of this large subcortical nucleus. In rodents, it is one nucleus penetrated by many fibers from the cerebral cortex. In larger animals and in humans, the area is split by a bundle of mainly corticofugal axons into the caudate nucleus and putamen. Dopamine input to both is similar and except for the details of cortical afferents to the two parts the striatum seems to act as one structure. Its main function is expected to be the transfer of the information carried in its cortical inputs onward through the basal ganglia. Diseases of this area of brain are associated with movement disorders and much is made of the action of dopamine on the long-term stability of corticostriatal synapses. The cortex is not at all the only input to the area, however, and the thalamus has almost as many synapses with striatal output neurons as has the cortex. This chapter summarizes the contributions to the study of the involvement of thalamostriatal inputs presented at Dopamine 2013 and emphasizes that this input, though largely ignored, has important lessons for those interested in understanding the function of the basal ganglia. 

Thalamostriatal synapses-another substrate for dopamine action?

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.

Extrasynaptic glutamate NMDA receptors: key players in striatal function.

The striosome (or patch) was first identified with anatomical techniques as neurons organized in a three-dimensional labyrinth inserted in and interdigitating the rest of neostriatum: the matrix. Striosome and matrix rapidly became known as two neuronal compartments expressing different biochemical markers, embryonic development and afferent and efferent connectivity. In spite of extensive intrinsic neuronal axonal and dendritic extensions supposed to exchange information between matrix and striosomes, evidence suggested the presence of independent areas. Here, we report that indeed these two areas do not exchange synaptic information. We used genetic expression of channel rhodopsin 2 carried by adeno-associated virus serotype 10 (AAVrh10) that only expresses in neurons of the matrix compartment. Whole-cell patch-clamp recordings of matrix neurons activated by light pulses consistently produced inhibitory postsynaptic currents (IPSCs), but the same manipulation did not evoke IPSCs in striosome neurons. The matrix contains both direct and indirect striatal output pathways. By targeting striatal matrix expression of designer receptors exclusively activated by a designer drug (DREADD) hM3di carried by AAVrh10, we were able to inhibit the matrix neuronal compartment of the dorsolateral striatum during performance of a learned single-pellet reach-to-grasp task. As expected, inhibition of matrix neurons by systemic administration of DREADD agonist clozapine-n-oxide interfered with performance of the learned task. 

The neostriatum: two entities, one structure?

Erratum to: The neostriatum: two entities, one structure?

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. 

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

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. 

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

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. 

Basal ganglia-thalamus and the "crowning enigma".

The aim of this work was to study release of glutamic acid (GLU) from one-axon terminal or bouton at-a-time using cortical neurons grown in vitro to study the effect of presynaptic auto- and heteroreceptor stimulation. Neurons were infected with release reporters SypHx2 or iGluSnFR at 7 or 3 days-in-vitro (DIV) respectively. At 13-15 DIV single synaptic boutons were identified from images obtained from a confocal scanning microscope before and after field electrical stimulation. We further stimulated release by raising intracellular levels of cAMP with forskolin (10µM). Forskolin-mediated effects were dependent on protein kinase A (PKA) and did not result from an increase in endocytosis, but rather from an increase in the size of the vesicle readily releasable pool. Once iGluSnFR was confirmed as more sensitive than SypHx2, it was used to study the participation of presynaptic auto- and heteroreceptors on GLU release. Although most receptor agonizts (carbamylcholine, nicotine, dopamine D2, BDNF) did not affect electrically stimulated GLU release, a significant increase was observed in the presence of metabotropic D1/D5 heteroreceptor agonist (SKF38393 10µM) that was reversed by PKA inhibitors. Interestingly, stimulation of group II metabotropic mGLU2/3 autoreceptors (LY379268 50nM) induced a decrease in GLU release that was reversed by the specific mGLU2/3 receptor antagonist (LY341495 1µM) and also by PKA inhibitors (KT5720 200nM and PKI14-22 400nM). These changes in release probability at individual release sites suggest another level of control of the distribution of transmitter substances in cortical tissue.

Presynaptic D1 heteroreceptors and mGlu autoreceptors act at individual cortical release sites to modify glutamate release.

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.

Cholinergic modulation of striatal microcircuits.

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.

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

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.

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

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.

Gordon W. Arbuthnott

Brain Mechanisms for Behaviour Unit

Okinawa Institute of Science and Technology Graduate University

In Vivo Wireless Optogenetic Control of Skilled Motor Behavior