Reversible inhibition of the basal ganglia prolongs repetitive vocalization but only weakly affects sequencing at branch points in songbirds.

Although vocal signals, including languages and songbird syllables, are composed of a finite number of acoustic elements, diverse vocal sequences are composed of a combination of these elements, which are linked together by syntactic rules. However, the neural basis of syntactic vocalization generation remains poorly understood. Here, we report that inhibition using tetrodotoxin (TTX) and manipulations of gamma-aminobutyric acid (GABA) receptors within the basal ganglia Area X or lateral magnocellular nucleus of the anterior neostriatum (LMAN) alter and prolong repetitive vocalization in Bengalese finches (Lonchura striata var. domestica). These results suggest that repetitive vocalizations are modulated by the basal ganglia and not solely by higher motor cortical neurons. These data highlight the importance of neural circuits, including the basal ganglia, in the production of stereotyped repetitive vocalizations and demonstrate that dynamic disturbances within the basal ganglia circuitry can differentially affect the repetitive temporal features of songs.

Cerebro-cerebellar interactions in nonhuman primates examined by optogenetic functional magnetic resonance imaging.

Functional magnetic resonance imaging (fMRI) is a promising approach for the simultaneous and extensive scanning of whole-brain activities. Optogenetics is free from electrical and magnetic artifacts and is an ideal stimulation method for combined use with fMRI. However, the application of optogenetics in nonhuman primates (NHPs) remains limited. Recently, we developed an efficient optogenetic intracortical microstimulation method of the primary motor cortex (M1), which successfully induced forelimb movements in macaque monkeys. Here, we aimed to investigate how optogenetic M1 stimulation causes neural modulation in the local and remote brain regions in anesthetized monkeys using 7-tesla fMRI. We demonstrated that optogenetic stimulation of the M1 forelimb and hindlimb regions successfully evoked robust direct and remote fMRI activities. Prominent remote activities were detected in the anterior and posterior lobes in the contralateral cerebellum, which receive projections polysynaptically from the M1. We further demonstrated that the cerebro-cerebellar projections from these M1 regions were topographically organized, which is concordant with the somatotopic map in the cerebellar cortex previously reported in macaques and humans. The present study significantly enhances optogenetic fMRI in NHPs, resulting in profound understanding of the brain network, thereby accelerating the translation of findings from animal models to humans.

Subthalamic nucleus stabilizes movements by reducing neural spike variability in monkey basal ganglia.

The subthalamic nucleus projects to the external and internal pallidum, the modulatory and output nuclei of the basal ganglia, respectively, and plays an indispensable role in controlling voluntary movements. However, the precise mechanism by which the subthalamic nucleus controls pallidal activity and movements remains elusive. Here, we utilize chemogenetics to reversibly reduce neural activity of the motor subregion of the subthalamic nucleus in three macaque monkeys (Macaca fuscata, both sexes) during a reaching task. Systemic administration of chemogenetic ligands prolongs movement time and increases spike train variability in the pallidum, but only slightly affects firing rate modulations. Across-trial analyses reveal that the irregular discharges in the pallidum coincides with prolonged movement time. Reduction of subthalamic activity also induces excessive abnormal movements in the contralateral forelimb, which are preceded by subthalamic and pallidal phasic activity changes. Our results suggest that the subthalamic nucleus stabilizes pallidal spike trains and achieves stable movements.

Resting-state dopaminergic cell firing in the ventral tegmental area negatively regulates affiliative social interactions in a developmental animal model of schizophrenia.

Hyperdopaminergic activities are often linked to positive symptoms of schizophrenia, but their neuropathological implications on negative symptoms are rather controversial among reports. Here, we explored the regulatory role of the resting state-neural activity of dopaminergic neurons in the ventral tegmental area (VTA) on social interaction using a developmental rat model for schizophrenia. We prepared the model by administering an ammonitic cytokine, epidermal growth factor (EGF), to rat pups, which later exhibit the deficits of social interaction as monitored with same-gender affiliative sniffing. In vivo single-unit recording and microdialysis revealed that the baseline firing frequency of and dopamine release from VTA dopaminergic neurons were chronically increased in EGF model rats, and their social interaction was concomitantly reduced. Subchronic treatment with risperidone ameliorated both the social interaction deficits and higher frequency of dopaminergic cell firing in this model. Sustained suppression of hyperdopaminergic cell firing in EGF model rats by DREADD chemogenetic intervention restored the event-triggered dopamine release and their social behaviors. These observations suggest that the higher resting-state activity of VTA dopaminergic neurons is responsible for the reduced social interaction of this schizophrenia model.

Abnormal Cortico-Basal Ganglia Neurotransmission in a Mouse Model of l-DOPA-Induced Dyskinesia.

l-3,4-dihydroxyphenylalanine (l-DOPA) is an effective treatment for Parkinson's disease (PD); however, long-term treatment induces l-DOPA-induced dyskinesia (LID). To elucidate its pathophysiology, we developed a mouse model of LID by daily administration of l-DOPA to PD male ICR mice treated with 6-hydroxydopamine (6-OHDA), and recorded the spontaneous and cortically evoked neuronal activity in the external segment of the globus pallidus (GPe) and substantia nigra pars reticulata (SNr), the connecting and output nuclei of the basal ganglia, respectively, in awake conditions. Spontaneous firing rates of GPe neurons were decreased in the dyskinesia-off state (≥24 h after l-DOPA injection) and increased in the dyskinesia-on state (20-100 min after l-DOPA injection while showing dyskinesia), while those of SNr neurons showed no significant changes. GPe and SNr neurons showed bursting activity and low-frequency oscillation in the PD, dyskinesia-off, and dyskinesia-on states. In the GPe, cortically evoked late excitation was increased in the PD and dyskinesia-off states but decreased in the dyskinesia-on state. In the SNr, cortically evoked inhibition was largely suppressed, and monophasic excitation became dominant in the PD state. Chronic l-DOPA treatment partially recovered inhibition and suppressed late excitation in the dyskinesia-off state. In the dyskinesia-on state, inhibition was further enhanced, and late excitation was largely suppressed. Cortically evoked inhibition and late excitation in the SNr are mediated by the cortico-striato-SNr direct and cortico-striato-GPe-subthalamo-SNr indirect pathways, respectively. Thus, in the dyskinesia-on state, signals through the direct pathway that release movements are enhanced, while signals through the indirect pathway that stop movements are suppressed, underlying LID.SIGNIFICANCE STATEMENT Parkinson's disease (PD) is caused by progressive loss of midbrain dopaminergic neurons, characterized by tremor, rigidity, and akinesia, and estimated to affect around six million people world-wide. Dopamine replacement therapy is the gold standard for PD treatment; however, control of symptoms using l-3,4-dihydroxyphenylalanine (l-DOPA) becomes difficult over time because of abnormal involuntary movements (AIMs) known as l-DOPA-induced dyskinesia (LID), one of the major issues for advanced PD. Our electrophysiological data suggest that dynamic changes in the basal ganglia circuitry underlie LID; signals through the direct pathway that release movements are enhanced, while signals through the indirect pathway that stop movements are suppressed. These results will provide the rationale for the development of more effective treatments for LID.

Weakly correlated activity of pallidal neurons in behaving monkeys.

The basal ganglia play a crucial role in the control of voluntary movements. Neurons in both the external and internal segments of the globus pallidus, the connecting and output nuclei of the basal ganglia, respectively, change their firing rates in relation to movements. Firing rate changes of movement-related neurons seem to convey signals for motor control. On the other hand, coincident spikes among neurons, that is, correlated activity, may also contribute to motor control. To address this issue, we first identified multiple pallidal neurons receiving inputs from the forelimb regions of the primary motor cortex and supplementary motor area, recorded neuronal activity of these neurons simultaneously, and analyzed their spike correlations while monkeys performed a hand-reaching task. Most (79%) pallidal neurons exhibited task-related firing rate changes, whereas only a small fraction (20%) showed significant but small and short correlated activity during the task performance. These results suggest that motor control signals are conveyed primarily by firing rate changes in the external and internal segments of the globus pallidus and that the contribution of correlated activity may play only a minor role in the healthy state.

All-solid-state ion-selective electrodes with redox-active lithium, sodium, and potassium insertion materials as the inner solid-contact layer.

All-solid-state ion-selective electrodes as potentiometric ion sensors for lithium, sodium, and potassium have been demonstrated by installing a composite layer containing a powder of alkali insertion materials, LixFePO4, Na0.33MnO2, and KxMnO2·nH2O, respectively, as an inner solid-contact layer between the electrode substrate and plasticized poly(vinyl chloride) (PVC)-based ion-sensitive membrane containing the corresponding ionophores for Li+, Na+, and K+ ions. These double-layer ion-selective electrodes, consisting of the composite and PVC layers prepared by a simple drop cast method, exhibit a quick potential response (less than 5 s) to each alkali-metal ion with sufficient Nernstian slopes of calibration curves, ca. 59 mV per decade. The installation of the insertion materials as the inner solid-contact layers is highly efficient for the stabilization of membrane potential, resulting in a prompt response to the alkali ion activity in the analyte, compared to those of the electrodes without the alkali insertion materials. From alternating current impedance measurements for the electrodes, the inner layer of the installed alkali insertion materials drastically reduces the impedance of the membrane/electrode interface, leading to an improvement in their ion-sensing performance.

A wireless neural recording system with a precision motorized microdrive for freely behaving animals.

The brain is composed of many different types of neurons. Therefore, analysis of brain activity with single-cell resolution could provide fundamental insights into brain mechanisms. However, the electrical signal of an individual neuron is very small, and precise isolation of single neuronal activity from moving subjects is still challenging. To measure single-unit signals in actively behaving states, establishment of technologies that enable fine control of electrode positioning and strict spike sorting is essential. To further apply such a single-cell recording approach to small brain areas in naturally behaving animals in large spaces or during social interaction, we developed a compact wireless recording system with a motorized microdrive. Wireless control of electrode placement facilitates the exploration of single neuronal activity without affecting animal behaviors. Because the system is equipped with a newly developed data-encoding program, the recorded data are readily compressed almost to theoretical limits and securely transmitted to a host computer. Brain activity can thereby be stably monitored in real time and further analyzed using online or offline spike sorting. Our wireless recording approach using a precision motorized microdrive will become a powerful tool for studying brain mechanisms underlying natural or social behaviors.

Genetic dissection of the circuit for hand dexterity in primates.

It is generally accepted that the direct connection from the motor cortex to spinal motor neurons is responsible for dexterous hand movements in primates. However, the role of the 'phylogenetically older' indirect pathways from the motor cortex to motor neurons, mediated by spinal interneurons, remains elusive. Here we used a novel double-infection technique to interrupt the transmission through the propriospinal neurons (PNs), which act as a relay of the indirect pathway in macaque monkeys (Macaca fuscata and Macaca mulatta). The PNs were double infected by injection of a highly efficient retrograde gene-transfer vector into their target area and subsequent injection of adeno-associated viral vector at the location of cell somata. This method enabled reversible expression of green fluorescent protein (GFP)-tagged tetanus neurotoxin, thereby permitting the selective and temporal blockade of the motor cortex­PN­motor neuron pathway. This treatment impaired reach and grasp movements, revealing a critical role for the PN-mediated pathway in the control of hand dexterity. Anti-GFP immunohistochemistry visualized the cell bodies and axonal trajectories of the blocked PNs, which confirmed their anatomical connection to motor neurons. This pathway-selective and reversible technique for blocking neural transmission does not depend on cell-specific promoters or transgenic techniques, and is a new and powerful tool for functional dissection in system-level neuroscience studies.

Neural coding of syntactic structure in learned vocalizations in the songbird.

Although vocal signals including human languages are composed of a finite number of acoustic elements, complex and diverse vocal patterns can be created from combinations of these elements, linked together by syntactic rules. To enable such syntactic vocal behaviors, neural systems must extract the sequence patterns from auditory information and establish syntactic rules to generate motor commands for vocal organs. However, the neural basis of syntactic processing of learned vocal signals remains largely unknown. Here we report that the basal ganglia projecting premotor neurons (HVC(X) neurons) in Bengalese finches represent syntactic rules that generate variable song sequences. When vocalizing an alternative transition segment between song elements called syllables, sparse burst spikes of HVC(X) neurons code the identity of a specific syllable type or a specific transition direction among the alternative trajectories. When vocalizing a variable repetition sequence of the same syllable, HVC(X) neurons not only signal the initiation and termination of the repetition sequence but also indicate the progress and state-of-completeness of the repetition. These different types of syntactic information are frequently integrated within the activity of single HVC(X) neurons, suggesting that syntactic attributes of the individual neurons are not programmed as a basic cellular subtype in advance but acquired in the course of vocal learning and maturation. Furthermore, some auditory-vocal mirroring type HVC(X) neurons display transition selectivity in the auditory phase, much as they do in the vocal phase, suggesting that these songbirds may extract syntactic rules from auditory experience and apply them to form their own vocal behaviors.