Kayo Nishizawa

Enhanced flexibility of place discrimination learning by targeting striatal cholinergic interneurons

Behavioural flexibility is mediated through the neural circuitry linking the prefrontal cortex and basal ganglia. Here we conduct selective elimination of striatal cholinergic interneurons in transgenic rats by immunotoxin-mediated cell targeting. Elimination of cholinergic interneurons from the dorsomedial striatum (DMS), but not from the dorsolateral striatum, results in enhanced reversal and extinction learning, sparing the acquisition of place discrimination. This enhancement is prevented by infusion of a non-selective muscarinic acetylcholine receptor agonist into the DMS either in the acquisition, reversal or extinction phase. In addition, gene-specific silencing of M4 muscarinic receptor by lentiviral expression of short hairpin RNA (shRNA) mimics the place reversal learning promoted by cholinergic elimination, whereas shRNA-mediated gene silencing of M1 muscarinic receptor shows the normal performance of reversal learning. Our data indicate that DMS cholinergic interneurons inhibit behavioural flexibility, mainly through the M4 muscarinic receptor, suggesting that this role is engaged to the stabilization of acquired reward contingency and the suppression of response switch to changed contingency.

Striatal Indirect Pathway Contributes to Selection Accuracy of Learned Motor Actions

The dorsal striatum, which contains the dorsolateral striatum (DLS) and dorsomedial striatum (DMS), integrates the acquisition and implementation of instrumental learning in cooperation with the nucleus accumbens (NAc). The dorsal striatum regulates the basal ganglia circuitry through direct and indirect pathways. The mechanism by which these pathways mediate the learning processes of instrumental actions remains unclear. We investigated how the striatal indirect (striatopallidal) pathway arising from the DLS contributes to the performance of conditional discrimination. Immunotoxin targeting of the striatal neuronal type containing dopamine D2 receptor in the DLS of transgenic rats resulted in selective, efficient elimination of the striatopallidal pathway. This elimination impaired the accuracy of response selection in a two-choice reaction time task dependent on different auditory stimuli. The impaired response selection was elicited early in the test sessions and was gradually restored as the sessions continued. The restoration from the deficits in auditory discrimination was prevented by excitotoxic lesion of the NAc but not by that of the DMS. In addition, lesion of the DLS mimicked the behavioral consequence of the striatopallidal removal at the early stage of test sessions of discriminative performance. Our results demonstrate that the DLS-derived striatopallidal pathway plays an essential role in the execution of conditional discrimination, showing its contribution to the control of selection accuracy of learned motor responses. The results also suggest the presence of a mechanism that compensates for the learning deficits during the repetitive sessions, at least partly, demanding accumbal function.

Striatal direct pathway modulates response time in execution of visual discrimination

The dorsal striatum in the basal ganglia circuitry is a principal structure that mediates the acquisition and performance of instrumental learning. The projections from the dorsal striatum are composed of two subpopulations of medium spiny neurons that constitute the direct and indirect pathways. The mechanism by which these striatal projections control the learning processes of instrumental actions remains unknown. We addressed the behavioral role of the striatal direct (striatonigral) pathway in the performance of visual discrimination. Immunotoxin targeting of the striatal neuronal type containing dopamine D1 receptor in mice resulted in a moderate level of elimination of the striatonigral pathway. Targeting of the neural pathway from the whole region of the dorsal striatum lengthened the response time but did not affect the accuracy of response selection in a two‐choice reaction time task dependent on light stimulus. This lengthened motor response was induced early in the test sessions and was gradually restored to normal levels during repetitive sessions. In addition, subregion‐specific pathway targeting revealed that the delay in learned motor response was generated by the elimination of the striatonigral pathway arising from the dorsomedial striatum but not from the dorsolateral striatum. Our findings indicate that the striatonigral pathway, in particular from the dorsomedial striatum, contributes to the regulation of response time in the execution of visual discrimination. The restoration of motor response deficits during repetitive sessions suggests the presence of a mechanism by which the response facilitation is acquired through continuation of learning despite the removal of the striatonigral pathway.

Distinct roles of basal forebrain cholinergic neurons in spatial and object recognition memory.

Recognition memory requires processing of various types of information such as objects and locations. Impairment in recognition memory is a prominent feature of amnesia and a symptom of Alzheimer’s disease (AD). Basal forebrain cholinergic neurons contain two major groups, one localized in the medial septum (MS)/vertical diagonal band of Broca (vDB), and the other in the nucleus basalis magnocellularis (NBM). The roles of these cell groups in recognition memory have been debated, and it remains unclear how they contribute to it. We use a genetic cell targeting technique to selectively eliminate cholinergic cell groups and then test spatial and object recognition memory through different behavioural tasks. Eliminating MS/vDB neurons impairs spatial but not object recognition memory in the reference and working memory tasks, whereas NBM elimination undermines only object recognition memory in the working memory task. These impairments are restored by treatment with acetylcholinesterase inhibitors, anti-dementia drugs for AD. Our results highlight that MS/vDB and NBM cholinergic neurons are not only implicated in recognition memory but also have essential roles in different types of recognition memory.

Differential roles of dopamine D1 and D2 receptor-containing neurons of the nucleus accumbens shell in behavioral sensitization

The nucleus accumbens (Nac) mediates the reinforcing and motor stimulating properties of psychostimulants. It receives dopaminergic afferents from the ventral midbrain and is divided into two distinct subregions: shell and core. Each of these contains two subtypes of medium spiny neurons, which express either dopamine D1 (D1R) or D2 (D2R) receptors. However, functional dissociation between the two subtypes in psychostimulant response remains to be elucidated. We performed selective ablation of each subtype in the Nac shell in mice, using immunotoxin‐mediated cell targeting, and examined the behavioral sensitization evoked by repeated administration of methamphetamine. The D1R cell‐ablated mice exhibited delayed induction of sensitized locomotion compared to control mice, whereas the D2R cell‐ablated mice showed a mildly enhanced rate of induction of sensitization. In vivo microdialysis revealed a marked blockade of the increase in extracellular dopamine in the Nac of the D1R cell‐ablated animals in response to methamphetamine, indicating that the observed delay in behavioral sensitization in these mice involves an impairment in accumbal dopamine release. Our results reveal differential roles of D1R‐ and D2R‐containing accumbal shell neurons in the development of behavioral sensitization to psychostimulants.

Lever pressing responses under a fixed-ratio schedule of mice with 6-hydroxydopamine-induced dopamine depletion in the nucleus accumbens.

In order to investigate the relationship between dopamine transmission in the nucleus accumbens and operant behavior in mice, mice with 6-hydroxydopamine (6-OHDA)-induced dopamine depletion in the nucleus accumbens were tested for their performance in lever pressing tasks under FR schedules with 8 ratios from FR5 to FR120. The mice were given one 20-mg food pellet per completed FR schedule in FR5, FR10, and FR20; they were given 2 pellets in FR40, and one more cumulatively in the rest of the schedules. Before the 6-OHDA injection surgery, all mice were trained to press a lever under all FR schedules. Then, 6-OHDA or ascorbate was injected into the nucleus accumbens. Postoperatively, the mice were tested under each FR schedule, with 3 sessions per schedule. 6-OHDA-treated mice exhibited an increase in lever pressing latency, i.e., the time interval between the last presentation of the reward and the next lever press, and a decrease in inter-response intervals, i.e., the time interval between 2 lever presses excluding lever pressing latency, irrespective of the FR ratios. Furthermore, in these 6-OHDA-treated mice, the number of lever presses during the first 300s of the session decreased under FR schedules with low ratios (5, 10, and 20). Open field activity, food motivation, and the amount of food consumed were not affected by dopamine depletion in the nucleus accumbens. These results suggest that the dopamine system in the nucleus accumbens had an important role in the control of lever pressing latency and inter-response intervals under FR reinforcement schedules.

Task-dependent function of striatal cholinergic interneurons in behavioural flexibility

Flexible switching of behaviours depends on integrative functioning through the neural circuit connecting the prefrontal cortex and the dorsomedial striatum (DMS). Although cholinergic interneurons modulate striatal outputs by diverse synaptic mechanisms, the roles of cholinergic interneurons in the DMS appear to vary among different models used to validate behavioural flexibility. Here, we conducted immunotoxin‐mediated cell targeting of DMS cholinergic interneurons and examined the functions of these interneurons in behavioural flexibility, with the learning conditions differing in trial spacing and discrimination type in a modified T‐maze. Elimination of the DMS cholinergic cell group normally spared reversal learning in place discrimination with an intertrial interval (ITI) of 15 s, but it impaired the reversal performance in response discrimination with the same ITI. In contrast, DMS cholinergic elimination resulted in enhanced reversal performance in both place and response discrimination tasks with a 10‐min ITI and accelerated the reversal of response discrimination with a 20‐min ITI. Our previous study also showed an enhanced influence of cholinergic targeting on place reversal learning with a 20‐min ITI, and the present results demonstrate that DMS cholinergic interneurons act to inhibit both place and response reversal performance with a relatively longer ITI, whereas their functions differ between types of reversal performance in the tasks with a shorter ITI. These findings suggest distinct roles of the DMS cholinergic cell group in behavioural flexibility dependent on the trial spacing and discrimination type constituting the learning tasks.

Action Selection and Flexible Switching Controlled by the Intralaminar Thalamic Neurons

Learning processes contributing to appropriate selection and flexible switching of behaviors are mediated through the dorsal striatum, a key structure of the basal ganglia circuit. The major inputs to striatal subdivisions are provided from the intralaminar thalamic nuclei, including the central lateral nucleus (CL) and parafascicular nucleus (PF). Thalamostriatal neurons in the PF modulate the acquisition and performance of stimulus-response learning. Here, we address the roles of the CL thalamostriatal neurons in learning processes by using a selective neural pathway targeting technique. We show that the CL neurons are essential for the performance of stimulus-response learning and for behavioral flexibility, including reversal and attentional set-shifting of learned responses. In addition, chemogenetic suppression of neural activity supports the requirements of these neurons for behavioral flexibility. Our results suggest that the main contribution of the CL thalamostriatal neurons is functional control of the basal ganglia circuit linked to the prefrontal cortex.

Targeted expression of step-function opsins in transgenic rats for optogenetic studies

Rats are excellent animal models for experimental neuroscience. However, the application of optogenetics in rats has been hindered because of the limited number of established transgenic rat strains. To accomplish cell-type specific targeting of an optimized optogenetic molecular tool, we generated ROSA26/CAG-floxed STOP-ChRFR(C167A)-Venus BAC rats that conditionally express the step-function mutant channelrhodopsin ChRFR(C167A) under the control of extrinsic Cre recombinase. In primary cultured cortical neurons derived from this reporter rat, only Cre-positive cells expressing ChRFR(C167A) became bi-stable, that is, their excitability was enhanced by blue light and returned to the baseline by yellow~red light. In bigenic pups carrying the Phox2B-Cre driver, ChRFR(C167A) was specifically expressed in the rostral parafacial respiratory group (pFRG) in the medulla, where endogenous Phox2b immunoreactivity was detected. These neurons were sensitive to blue light with an increase in the firing frequency. Thus, this transgenic rat actuator/reporter system should facilitate optogenetic studies involving the effective in vivo manipulation of the activities of specific cell fractions using light of minimal intensity.

Neural Circuit Mechanism for Learning Dependent on Dopamine Transmission: Roles of Striatal Direct and Indirect Pathways in Sensory Discrimination

The dorsal striatum in basal ganglia circuit mediates learning processes contributing to instrumental motor actions. The striatum receives excitatory inputs from many cortical areas and the thalamic nuclei and dopaminergic inputs from the ventral midbrain and projects to the output nuclei through direct and indirect pathways. The neural mechanism remains unclear as to how these striatofugal pathways control the learning processes of instrumental actions. Here, we addressed the behavioral roles of the two striatofugal pathways in the performance of sensory discrimination by using immunotoxin (IT)-mediated cell targeting. IT targeting of the striatal direct pathway in mutant mice lengthened the response time but did not affect the accuracy of the response selection in visual discrimination. Subregion-specific pathway targeting revealed a delay in motor responses generated by elimination of the direct pathway arising from the dorsomedial striatum (DMS) but not from the dorsolateral striatum (DLS). These findings indicate that the direct pathway, in particular that from the DMS, contributes to the regulation of the response time in visual discrimination. In addition, IT targeting of the striatal indirect pathway originating from the DLS in transgenic rats impaired the accuracy of response selection in auditory discrimination, whereas the response time remained normal. These data demonstrate that the DLS-derived indirect pathway plays an essential role in the control of the selection accuracy of learned motor responses. Our results suggest that striatal direct and indirect pathways act cooperatively to regulate the selection accuracy and response time of learned motor actions in the performance of discriminative learning.