Phasic dopamine signals are reduced in the spontaneously hypertensive rat and increased by methylphenidate.

The spontaneously hypertensive rat (SHR) is a selectively bred animal strain that is frequently used to model attention-deficit hyperactivity disorder (ADHD) because of certain genetically determined behavioural characteristics. To test the hypothesis that the characteristically altered response to positive reinforcement in SHRs may be due to altered phasic dopamine response to reward, we measured phasic dopamine signals in the SHRs and Sprague Dawley (SD) rats using in vivo fast-scan cyclic voltammetry. The effects of the dopamine reuptake inhibitor, methylphenidate, on these signals were also studied. Phasic dopamine signals during the pairing of a sensory cue with electrical stimulation of midbrain dopamine neurons were significantly smaller in the SHRs than in the SD rats. Over repeated pairings, the dopamine response to the sensory cue increased, whereas the response to the electrical stimulation of dopamine neurons decreased, similarly in both strains. However, the final amplitude of the response to the sensory cue after pairing was significantly smaller in SHRs than in the SD rats. Methylphenidate increased responses to sensory cues to a significantly greater extent in the SHRs than in the SD rats, due largely to differences in the low dose effect. At a higher dose, methylphenidate increased responses to sensory cues and electrical stimulation similarly in SHRs and SD rats. The smaller dopamine responses may explain the reduced salience of reward-predicting cues previously reported in the SHR, whereas the action of methylphenidate on the cue response suggests a potential mechanism for the therapeutic effects of low-dose methylphenidate in ADHD.

Acetylcholine waves and dopamine release in the striatum.

Striatal dopamine encodes reward, with recent work showing that dopamine release occurs in spatiotemporal waves. However, the mechanism of dopamine waves is unknown. Here we report that acetylcholine release in mouse striatum also exhibits wave activity, and that the spatial scale of striatal dopamine release is extended by nicotinic acetylcholine receptors. Based on these findings, and on our demonstration that single cholinergic interneurons can induce dopamine release, we hypothesized that the local reciprocal interaction between cholinergic interneurons and dopamine axons suffices to drive endogenous traveling waves. We show that the morphological and physiological properties of cholinergic interneuron - dopamine axon interactions can be modeled as a reaction-diffusion system that gives rise to traveling waves. Analytically-tractable versions of the model show that the structure and the nature of propagation of acetylcholine and dopamine traveling waves depend on their coupling, and that traveling waves can give rise to empirically observed correlations between these signals. Thus, our study provides evidence for striatal acetylcholine waves in vivo, and proposes a testable theoretical framework that predicts that the observed dopamine and acetylcholine waves are strongly coupled phenomena.

Conditioned approach behavior of SHR and SD rats during Pavlovian conditioning.

Individual differences in reward-related learning are relevant to many behavioral disorders. Sensory cues that predict reward can become incentive stimuli that adaptively support behavior, or alternatively, cause maladaptive behaviors. The spontaneously hypertensive rat (SHR) expresses a genetically determined elevated sensitivity to delay of reward, and has been extensively studied as a behavioral model for attention deficit hyperactivity disorder (ADHD). We investigated reward-related learning in the SHR, comparing them to Sprague-Dawley (SD) rats as a reference strain. A standard Pavlovian conditioned approach task was used, in which a lever cue was followed by reward. Lever presses could occur while the lever was extended, but had no effect on reward delivery. The behavior of both the SHRs and the SD rats showed that they learnt that the lever cue predicted reward. However, the pattern of behavior differed between the strains. During lever cue presentation, SD rats pressed the lever more often and made fewer magazine entries than SHRs. When lever contacts that did not result in lever presses were analyzed, there was no significant difference between SHRs and SDs. These results suggest that the SHRs attributed less incentive value to the conditioned stimulus than the SD rats. During the presentation of the conditioned cue, cue directed responses are called sign tracking responses, whereas responses directed towards the food magazine are called goal tracking responses. Analysis of behavior using a standard Pavlovian conditioned approach index to quantify sign and goal tracking tendencies showed that both strains had a tendency towards goal tracking in this task. However, the SHRs showed a significantly greater goal tracking tendency than the SD rats. Taken together, these findings suggest that attribution of incentive value to reward predicting cues is attenuated in SHRs, which might explain their elevated sensitivity to delay of reward.

Three-Dimensional Spatial Analyses of Cholinergic Neuronal Distributions Across The Mouse Septum, Nucleus Basalis, Globus Pallidus, Nucleus Accumbens, and Caudate-Putamen.

Neuronal networks are regulated by three-dimensional spatial and structural properties. Despite robust evidence of functional implications in the modulation of cognition, little is known about the three-dimensional internal organization of cholinergic networks in the forebrain. Cholinergic networks in the forebrain primarily occur in subcortical nuclei, specifically the septum, nucleus basalis, globus pallidus, nucleus accumbens, and the caudate-putamen. Therefore, the present investigation analyzed the three-dimensional spatial organization of 14,000 cholinergic neurons that expressed choline acetyltransferase (ChAT) in these subcortical nuclei of the mouse forebrain. Point process theory and graph signal processing techniques identified three topological principles of organization. First, cholinergic interneuronal distance is not uniform across brain regions. Specifically, in the septum, globus pallidus, nucleus accumbens, and the caudate-putamen, the cholinergic neurons were clustered compared with a uniform random distribution. In contrast, in the nucleus basalis, the cholinergic neurons had a spatial distribution of greater regularity than a uniform random distribution. Second, a quarter of the caudate-putamen is composed of axonal bundles, yet the spatial distribution of cholinergic neurons remained clustered when axonal bundles were accounted for. However, comparison with an inhomogeneous Poisson distribution showed that the nucleus basalis and caudate-putamen findings could be explained by density gradients in those structures. Third, the number of cholinergic neurons varies as a function of the volume of a specific brain region but cell body volume is constant across regions. The results of the present investigation provide topographic descriptions of cholinergic somata distribution and axonal conduits, and demonstrate spatial differences in cognitive control networks. The study provides a comprehensive digital database of the total population of ChAT-positive neurons in the reported structures, with the x,y,z coordinates of each neuron at micrometer resolution. This information is important for future digital cellular atlases and computational models of the forebrain cholinergic system enabling models based on actual spatial geometry.

Coincidence of cholinergic pauses, dopaminergic activation and depolarisation of spiny projection neurons drives synaptic plasticity in the striatum.

Dopamine-dependent long-term plasticity is believed to be a cellular mechanism underlying reinforcement learning. In response to reward and reward-predicting cues, phasic dopamine activity potentiates the efficacy of corticostriatal synapses on spiny projection neurons (SPNs). Since phasic dopamine activity also encodes other behavioural variables, it is unclear how postsynaptic neurons identify which dopamine event is to induce long-term plasticity. Additionally, it is unknown how phasic dopamine released from arborised axons can potentiate targeted striatal synapses through volume transmission. To examine these questions we manipulated striatal cholinergic interneurons (ChIs) and dopamine neurons independently in two distinct in vivo paradigms. We report that long-term potentiation (LTP) at corticostriatal synapses with SPNs is dependent on the coincidence of pauses in ChIs and phasic dopamine activation, critically accompanied by SPN depolarisation. Thus, the ChI pause defines the time window for phasic dopamine to induce plasticity, while depolarisation of SPNs constrains the synapses eligible for plasticity.

Striatal Cholinergic Signaling in Time and Space.

The cholinergic interneurons of the striatum account for a small fraction of all striatal cell types but due to their extensive axonal arborization give the striatum the highest content of acetylcholine of almost any nucleus in the brain. The prevailing theory of striatal cholinergic interneuron signaling is that the numerous varicosities on the axon produce an extrasynaptic, volume-transmitted signal rather than mediating rapid point-to-point synaptic transmission. We review the evidence for this theory and use a mathematical model to integrate the measurements reported in the literature, from which we estimate the temporospatial distribution of acetylcholine after release from a synaptic vesicle and from multiple vesicles during tonic firing and pauses. Our calculations, together with recent data from genetically encoded sensors, indicate that the temporospatial distribution of acetylcholine is both short-range and short-lived, and dominated by diffusion. These considerations suggest that acetylcholine signaling by cholinergic interneurons is consistent with point-to-point transmission within a steep concentration gradient, marked by transient peaks of acetylcholine concentration adjacent to release sites, with potential for faithful transmission of spike timing, both bursts and pauses, to the postsynaptic cell. Release from multiple sites at greater distance contributes to the ambient concentration without interference with the short-range signaling. We indicate several missing pieces of evidence that are needed for a better understanding of the nature of synaptic transmission by the cholinergic interneurons of the striatum.

An On-Demand Drug Delivery System for Control of Epileptiform Seizures.

Drug delivery systems have the potential to deliver high concentrations of drug to target areas on demand, while elsewhere and at other times encapsulating the drug, to limit unwanted actions. Here we show proof of concept in vivo and ex vivo tests of a novel drug delivery system based on hollow-gold nanoparticles tethered to liposomes (HGN-liposomes), which become transiently permeable when activated by optical or acoustic stimulation. We show that laser or ultrasound simulation of HGN-liposomes loaded with the GABAA receptor agonist, muscimol, triggers rapid and repeatable release in a sufficient concentration to inhibit neurons and suppress seizure activity. In particular, laser-stimulated release of muscimol from previously injected HGN-liposomes caused subsecond hyperpolarizations of the membrane potential of hippocampal pyramidal neurons, measured by whole cell intracellular recordings with patch electrodes. In hippocampal slices and hippocampal-entorhinal cortical wedges, seizure activity was immediately suppressed by muscimol release from HGN-liposomes triggered by laser or ultrasound pulses. After intravenous injection of HGN-liposomes in whole anesthetized rats, ultrasound stimulation applied to the brain through the dura attenuated the seizure activity induced by pentylenetetrazol. Ultrasound alone, or HGN-liposomes without ultrasound stimulation, had no effect. Intracerebrally-injected HGN-liposomes containing kainic acid retained their contents for at least one week, without damage to surrounding tissue. Thus, we demonstrate the feasibility of precise temporal control over exposure of neurons to the drug, potentially enabling therapeutic effects without continuous exposure. For future application, studies on the pharmacokinetics, pharmacodynamics, and toxicity of HGN-liposomes and their constituents, together with improved methods of targeting, are needed, to determine the utility and safety of the technology in humans.

The Effect of Serotonin Receptor 5-HT1B on Lateral Inhibition between Spiny Projection Neurons in the Mouse Striatum.

The principal neurons of the striatum, the spiny projection neurons (SPNs), make inhibitory synaptic connections with each other via collaterals of their main axon, forming a local lateral inhibition network. Serotonin, acting via the 5-HT1B receptor, modulates neurotransmitter release from SPN terminals in striatal output nuclei, but the role of 5-HT1B receptors in lateral inhibition among SPNs in the striatum is unknown. Here, we report the effects of 5-HT1B receptor activation on lateral inhibition in the mouse striatum. Whole-cell recordings were made from SPNs in acute brain slices of either sex, while optogenetically activating presynaptic SPNs or fast-spiking interneurons (FSIs). Activation of 5-HT1B receptors significantly reduced the amplitude of IPSCs evoked by optical stimulation of both direct and indirect pathway SPNs. This reduction was blocked by application of a 5-HT1B receptor antagonist. Activation of 5-HT1B receptors did not reduce the amplitude of IPSCs evoked from FSIs. These results suggest a new role for serotonin as a modulator of lateral inhibition among striatal SPNs. The 5-HT1B receptor may, therefore, be a suitable target for future behavioral experiments investigating the currently unknown role of lateral inhibition in the function of the striatum.SIGNIFICANCE STATEMENT We show that stimulation of serotonin receptors reduces the efficacy of lateral inhibition between spiny projection neurons (SPNs), one of the biggest GABAergic sources in the striatum, by activation of the serotonin 5-HT1B receptor. The striatum receives serotonergic input from the dorsal raphe nuclei and is important in behavioral brain functions like learning and action selection. Our findings suggest a new role for serotonin in modulating the dynamics of neural interactions in the striatum, which extends current knowledge of the mechanisms of the behavioral effects of serotonin.

Behavioral determinants in the expression of the Kamin blocking effect: Implications for associative learning theory.

Associative learning makes important contributions to our behavior and decisions. The Kamin blocking effect is an associative learning phenomenon that plays a central role in understanding of the psychological principles underlying associative learning. However, several recent failures to replicate the blocking effect suggest that the conditions necessary for blocking are poorly understood. To understand the conditions necessary for blocking, here we review studies into the expression of blocking in subjects that either approach and interact with the conditioned cue (sign trackers) or approach and interact with the reward location (goal trackers) during appetitive classical conditioning. Psychological theory and the neurophysiological correlates of appetitive classical conditioning make opposing predictions regarding the expression of blocking in sign and goal trackers. We reconcile these opposing predictions in a qualitative model using two parallel learning processes. Such models offer a better framework for understanding the psychological associative structures acquired during learning, their interactions contributing to the conditioned response, and how they affect subsequent learning and the expression of the Kamin blocking effect.

Responses of putative medium spiny neurons and fast-spiking interneurons to reward-related sensory signals in Wistar and genetically hypertensive rats.

Medium spiny neurons (MSN) are the primary output neurons of the striatum. Their activity is modulated by exogenous afferents and local circuit inputs, including fast-spiking interneurons (FSI). Altered responses of MSN and FSI may account for altered reward-driven behaviour in hyperactive rat strains, such as the genetically hypertensive (GH) rat. To investigate whether striatal neuron responses differ between GH and Wistar rats, we recorded putative MSNs (pMSN) and FSI (pFSI) from freely moving GH and Wistar rats in a classically conditioned (Pavlovian) cue-reward association paradigm. Here, the same auditory cue signal predicted reward delivery in one block of trials, but was not followed by reward in another. The significance of the cue as a reward predictor was indicated during each block by an environmental context provided by the house light. The results showed that pMSN in GH rats, but not Wistar rats, were more sensitive to the auditory signal in the context indicating no-reward, than in the reward context. Such enhanced sensitivity to cues in a no-reward context may contribute to a specific deficit in instrumental behaviour seen in GH rats, which maintain higher levels of instrumental responding in a context that indicates responding will not be rewarded. In addition, pFSI also responded to auditory signals, but there was no significant effect of reward context. Surprisingly, given their known feed-forward role, pFSI responded at longer latency than pMSN, suggesting that relative timing of activity in the two populations may be task specific.

The nucleus accumbens and inhibition in the ventral tegmental area play a causal role in the Kamin blocking effect.

The discovery of Kamin blocking led to the idea that associative learning occurs only when there is a mismatch between actual and predicted outcomes, or prediction error. The neural substrates involved in regulating this prediction error during behavioral learning are still not fully elucidated. We investigated in rats the role of the ventral tegmental area and the nucleus accumbens in Kamin blocking. Our blocking paradigm involved three phases: appetitive classical conditioning of a lever cue, conditioning of a compound of the lever cue plus an auditory cue, and testing response to the auditory cue in extinction. We found that disruption of inhibition in the ventral tegmental area by bicuculline, or designer receptor mediated inactivation of the nucleus accumbens, during compound cue conditioning, attenuated Kamin blocking. These results suggest that inhibition in the ventral tegmental area and inhibitory output from the nucleus accumbens are necessary for blocking and make behaviorally significant contributions to the computation of reward prediction error. In addition, we found that inactivating the neurons in the nucleus accumbens during classical conditioning of the lever cue also attenuated blocking, without affecting classical conditioning of the lever. This indicates that learning in the nucleus accumbens is necessary for blocking and reward estimation. Our results reveal a causal role for nucleus accumbens modulated inhibitory inputs to the ventral tegmental area in the blocking effect and suggest that they contribute to computation of reward prediction error during associative learning.

Multiparametric assessment of the impact of opsin expression and anesthesia on striatal cholinergic neurons and auditory brainstem activity.

Recent developments in genetic engineering have established murine models that permit the selective control of cholinergic neurons via optical stimulation. Despite copious benefits granted by these experimental advances, the sensory physiognomy of these organisms has remained poorly understood. Therefore, the present study evaluates sensory and neuronal response properties of animal models developed for the study of optically induced acetylcholine release regulation. Auditory brainstem responses, fluorescence imaging, and patch clamp recording techniques were used to assess the impact of viral infection, sex, age, and anesthetic agents across the ascending auditory pathway of ChAT-Cre and ChAT-ChR2(Ai32) mice. Data analyses revealed that neither genetic configuration nor adeno-associated viral infection alters the early stages of auditory processing or the cellular response properties of cholinergic neurons. However, anesthetic agent and dosage amount profoundly modulate the response properties of brainstem neurons. Last, analyses of age-related hearing loss in virally infected ChAT-Cre mice did not differ from those reported in wild type animals. This investigation demonstrates that ChAT-Cre and ChAT-ChR2(Ai32) mice are viable models for the study of cholinergic modulation in auditory processing, and it emphasizes the need for prudence in the selection of anesthetic procedures.

An open cortico-basal ganglia loop allows limbic control over motor output via the nigrothalamic pathway.

Cortico-basal ganglia-thalamocortical loops are largely conceived as parallel circuits that process limbic, associative, and sensorimotor information separately. Whether and how these functionally distinct loops interact remains unclear. Combining genetic and viral approaches, we systemically mapped the limbic and motor cortico-basal ganglia-thalamocortical loops in rodents. Despite largely closed loops within each functional domain, we discovered a unidirectional influence of the limbic over the motor loop via ventral striatum-substantia nigra (SNr)-motor thalamus circuitry. Slice electrophysiology verifies that the projection from ventral striatum functionally inhibits nigro-thalamic SNr neurons. In vivo optogenetic stimulation of ventral or dorsolateral striatum to SNr pathway modulates activity in medial prefrontal cortex (mPFC) and motor cortex (M1), respectively. However, whereas the dorsolateral striatum-SNr pathway exerts little impact on mPFC, activation of the ventral striatum-SNr pathway effectively alters M1 activity. These results demonstrate an open cortico-basal ganglia loop whereby limbic information could modulate motor output through ventral striatum control of M1.

Role of homeostatic feedback mechanisms in modulating methylphenidate actions on phasic dopamine signaling in the striatum of awake behaving rats.

Methylphenidate is an established treatment for attention-deficit hyperactivity disorder that also has abuse potential. Both properties may relate to blocking dopamine and norepinephrine reuptake. We measured the effects of methylphenidate on dopamine dynamics in freely moving rats. Methylphenidate alone had no effect on the amplitude of phasic responses to cues or reward. However, when administered with the D2 receptor antagonist raclopride, methylphenidate increased dopamine responses, while raclopride alone had no effect. Using brain slices of substantia nigra or striatum, we confirmed that methylphenidate effects on firing rate of nigral dopamine neurons and dopamine release from terminals are constrained by negative feedback. A computational model using physiologically relevant parameters revealed that actions of methylphenidate on norepinephrine and dopamine transporters, and the effects of changes in tonic dopamine levels on D2 receptors, are necessary and sufficient to account for the experimental findings. In addition, non-linear fitting of the model to the data from freely moving animals revealed that methylphenidate significantly slowed the initial cue response dynamics. These results show that homeostatic regulation of dopamine release in the face of changing tonic levels of extracellular dopamine should be taken into account to understand the therapeutic benefits and abuse potential of methylphenidate.

Active propagation of dendritic electrical signals in C. elegans.

Active propagation of electrical signals in C. elegans neurons requires ion channels capable of regenerating membrane potentials. Here we report regenerative depolarization of a major gustatory sensory neuron, ASEL. Whole-cell patch-clamp recordings in vivo showed supralinear depolarization of ASEL upon current injection. Furthermore, stimulation of animal's nose with NaCl evoked all-or-none membrane depolarization in ASEL. Mutant analysis showed that EGL-19, the α1 subunit of L-type voltage-gated Ca2+ channels, is essential for regenerative depolarization of ASEL. ASEL-specific knock-down of EGL-19 by RNAi demonstrated that EGL-19 functions in C. elegans chemotaxis along an NaCl gradient. These results demonstrate that a natural substance induces regenerative all-or-none electrical signals in dendrites, and that these signals are essential for activation of sensory neurons for chemotaxis. As in other vertebrate and invertebrate nervous systems, active information processing in dendrites occurs in C. elegans, and is necessary for adaptive behavior.

Cholinergic interneurons in the rat striatum modulate substitution of habits.

Behavioural flexibility is crucial for adaptive behaviour, and recent evidence suggests that cholinergic interneurons of the striatum play a distinct role. Previous studies of cholinergic function have focused on strategy switching by the dorsomedial or ventral striatum. We here investigated whether cholinergic interneurons in the dorsolateral striatum play a similar role at the level of switching of habitual responses. Because the dorsolateral striatum is particularly involved in habitual responding, we developed a habit substitution task that involved switching habitual lever-press responses to one side to another. We first measured the effect of cholinergic activation in the dorsolateral striatum on this task. Chemogenetic activation of cholinergic interneurons caused an increase in the response rate for the substituted response that was significantly greater than the increase normally seen in control animals. The increase was due to burst-like responses with shorter inter-press intervals. However, there was no effect on inhibiting the old habit, or on habitual responding that did not require a switch. There was also no effect on lever-press performance and its reversal before lever-press responses became habitual. Conversely, neurochemically specific ablation of cholinergic interneurons did not significantly change habitual responding or response substitution. Thus, activation -but not ablation -of cholinergic interneurons in the dorsolateral striatum modulates expression of a new habit when an old habit is replaced by a new one. Together with previous work, this suggests that striatal cholinergic interneurons facilitate behavioural flexibility in both dorsolateral striatum in addition to dorsomedial and ventral striatum.

New Variations for Strategy Set-shifting in the Rat.

Behavioral flexibility is crucial for survival in changing environments. Broadly defined, behavioral flexibility requires a shift of behavioral strategy based on a change in governing rules. We describe a strategy set-shifting task that requires an attentional shift from one stimulus dimension to another. The paradigm is often used for testing cognitive flexibility in primates. However, the rodent version has not been as extensively developed. We have recently extended an established set-shifting task in the rat1 by requiring attention to different stimuli according to context. All the experimental conditions required animals to choose either a left or right lever. Initially, all animals had to choose on the basis of the location of the lever. Subsequently, a change in the rule occurred, which required a shift in set from location-based rule to a rule in which the correct lever was indicated by a light cue. We compared performance on three different versions of the task, in which the light stimulus was either novel, previously relevant, or previously irrelevant. We found that specific neurochemical lesions selectively impaired the ability to make particular types of set shift as measured by the performance on the different versions of the task.

A Streamlined Method for the Preparation of Gelatin Embedded Brains and Simplified Organization of Sections for Serial Reconstructions.

Gelatin embedding of whole brains for sectioning is a critical procedure used in neuroscience to ensure all morphological and spatial details are preserved intact. Here, we describe an inexpensive, reproducible and efficient means to embed post-fixed brains ready for sectioning in gelatin within a week's time. The sections obtained are distortion-free and their fragile internal structures preserved which can be used for serial reconstructions for lesion studies and mapping of viral expression after stereotaxic injections. In addition, the separation of adjacent slices into a series of 3-4 vials facilitates subsequent organization and assembly of serial sections at the mounting step.

Role of Striatal Cholinergic Interneurons in Set-Shifting in the Rat.

The ability to change strategies in different contexts is a form of behavioral flexibility that is crucial for adaptive behavior. The striatum has been shown to contribute to certain forms of behavioral flexibility such as reversal learning. Here we report on the contribution of striatal cholinergic interneurons-a key element in the striatal neuronal circuit-to strategy set-shifting in which an attentional shift from one stimulus dimension to another is required. We made lesions of rat cholinergic interneurons in dorsomedial or ventral striatum using a specific immunotoxin and investigated the effects on set-shifting paradigms and on reversal learning. In shifting to a set that required attention to a previously irrelevant cue, lesions of dorsomedial striatum significantly increased the number of perseverative errors. In this condition, the number of never-reinforced errors was significantly decreased in both types of lesions. When shifting to a set that required attention to a novel cue, rats with ventral striatum lesions made more perseverative errors. Neither lesion impaired learning of the initial response strategy nor a subsequent switch to a new strategy when response choice was indicated by a previously relevant cue. Reversal learning was not affected. These results suggest that in set-shifting the striatal cholinergic interneurons play a fundamental role, which is dissociable between dorsomedial and ventral striatum depending on behavioral context. We propose a common mechanism in which cholinergic interneurons inhibit neurons representing the old strategy and enhance plasticity underlying exploration of a new rule.