Representation of spontaneous movement by dopaminergic neurons is cell-type selective and disrupted in parkinsonism.

Midbrain dopaminergic neurons are essential for appropriate voluntary movement, as epitomized by the cardinal motor impairments arising in Parkinson's disease. Understanding the basis of such motor control requires understanding how the firing of different types of dopaminergic neuron relates to movement and how this activity is deciphered in target structures such as the striatum. By recording and labeling individual neurons in behaving mice, we show that the representation of brief spontaneous movements in the firing of identified midbrain dopaminergic neurons is cell-type selective. Most dopaminergic neurons in the substantia nigra pars compacta (SNc), but not in ventral tegmental area or substantia nigra pars lateralis, consistently represented the onset of spontaneous movements with a pause in their firing. Computational modeling revealed that the movement-related firing of these dopaminergic neurons can manifest as rapid and robust fluctuations in striatal dopamine concentration and receptor activity. The exact nature of the movement-related signaling in the striatum depended on the type of dopaminergic neuron providing inputs, the striatal region innervated, and the type of dopamine receptor expressed by striatal neurons. Importantly, in aged mice harboring a genetic burden relevant for human Parkinson's disease, the precise movement-related firing of SNc dopaminergic neurons and the resultant striatal dopamine signaling were lost. These data show that distinct dopaminergic cell types differentially encode spontaneous movement and elucidate how dysregulation of their firing in early Parkinsonism can impair their effector circuits.

LRRK2 BAC transgenic rats develop progressive, L-DOPA-responsive motor impairment, and deficits in dopamine circuit function.

Mutations in leucine-rich repeat kinase 2 (LRRK2) lead to late-onset, autosomal dominant Parkinson's disease, characterized by the degeneration of dopamine neurons of the substantia nigra pars compacta, a deficit in dopamine neurotransmission and the development of motor and non-motor symptoms. The most prevalent Parkinson's disease LRRK2 mutations are located in the kinase (G2019S) and GTPase (R1441C) encoding domains of LRRK2. To better understand the sequence of events that lead to progressive neurophysiological deficits in vulnerable neurons and circuits in Parkinson's disease, we have generated LRRK2 bacterial artificial chromosome transgenic rats expressing either G2019S or R1441C mutant, or wild-type LRRK2, from the complete human LRRK2 genomic locus, including endogenous promoter and regulatory regions. Aged (18-21 months) G2019S and R1441C mutant transgenic rats exhibit L-DOPA-responsive motor dysfunction, impaired striatal dopamine release as determined by fast-scan cyclic voltammetry, and cognitive deficits. In addition, in vivo recordings of identified substantia nigra pars compacta dopamine neurons in R1441C LRRK2 transgenic rats reveal an age-dependent reduction in burst firing, which likely results in further reductions to striatal dopamine release. These alterations to dopamine circuit function occur in the absence of neurodegeneration or abnormal protein accumulation within the substantia nigra pars compacta, suggesting that nigrostriatal dopamine dysfunction precedes detectable protein aggregation and cell death in the development of Parkinson's disease. In conclusion, our longitudinal deep-phenotyping provides novel insights into how the genetic burden arising from human mutant LRRK2 manifests as early pathophysiological changes to dopamine circuit function and highlights a potential model for testing Parkinson's therapeutics.

Deficits in dopaminergic transmission precede neuron loss and dysfunction in a new Parkinson model.

The pathological end-state of Parkinson disease is well described from postmortem tissue, but there remains a pressing need to define early functional changes to susceptible neurons and circuits. In particular, mechanisms underlying the vulnerability of the dopamine neurons of the substantia nigra pars compacta (SNc) and the importance of protein aggregation in driving the disease process remain to be determined. To better understand the sequence of events occurring in familial and sporadic Parkinson disease, we generated bacterial artificial chromosome transgenic mice (SNCA-OVX) that express wild-type α-synuclein from the complete human SNCA locus at disease-relevant levels and display a transgene expression profile that recapitulates that of endogenous α-synuclein. SNCA-OVX mice display age-dependent loss of nigrostriatal dopamine neurons and motor impairments characteristic of Parkinson disease. This phenotype is preceded by early deficits in dopamine release from terminals in the dorsal, but not ventral, striatum. Such neurotransmission deficits are not seen at either noradrenergic or serotoninergic terminals. Dopamine release deficits are associated with an altered distribution of vesicles in dopaminergic axons in the dorsal striatum. Aged SNCA-OVX mice exhibit reduced firing of SNc dopamine neurons in vivo measured by juxtacellular recording of neurochemically identified neurons. These progressive changes in vulnerable SNc neurons were observed independently of overt protein aggregation, suggesting neurophysiological changes precede, and are not driven by, aggregate formation. This longitudinal phenotyping strategy in SNCA-OVX mice thus provides insights into the region-specific neuronal disturbances preceding and accompanying Parkinson disease.

Cortical and thalamic excitation mediate the multiphasic responses of striatal cholinergic interneurons to motivationally salient stimuli.

Cholinergic interneurons are key components of striatal microcircuits. In primates, tonically active neurons (putative cholinergic interneurons) exhibit multiphasic responses to motivationally salient stimuli that mirror those of midbrain dopamine neurons and together these two systems mediate reward-related learning in basal ganglia circuits. Here, we addressed the potential contribution of cortical and thalamic excitatory inputs to the characteristic multiphasic responses of cholinergic interneurons in vivo. We first recorded and labeled individual cholinergic interneurons in anesthetized rats. Electron microscopic analyses of these labeled neurons demonstrated that an individual interneuron could form synapses with cortical and, more commonly, thalamic afferents. Single-pulse electrical stimulation of ipsilateral frontal cortex led to robust short-latency (<20 ms) interneuron spiking, indicating monosynaptic connectivity, but firing probability progressively decreased during high-frequency pulse trains. In contrast, single-pulse thalamic stimulation led to weak short-latency spiking, but firing probability increased during pulse trains. After initial excitation from cortex or thalamus, interneurons displayed a "pause" in firing, followed by a "rebound" increase in firing rate. Across all stimulation protocols, the number of spikes in the initial excitation correlated positively with pause duration and negatively with rebound magnitude. The magnitude of the initial excitation, therefore, partly determined the profile of later components of multiphasic responses. Upon examining the responses of tonically active neurons in behaving primates, we found that these correlations held true for unit responses to a reward-predicting stimulus, but not to the reward alone, delivered outside of any task. We conclude that excitatory inputs determine, at least in part, the multiphasic responses of cholinergic interneurons under specific behavioral conditions.

A major external source of cholinergic innervation of the striatum and nucleus accumbens originates in the brainstem.

Cholinergic transmission in the striatal complex is critical for the modulation of the activity of local microcircuits and dopamine release. Release of acetylcholine has been considered to originate exclusively from a subtype of striatal interneuron that provides widespread innervation of the striatum. Cholinergic neurons of the pedunculopontine (PPN) and laterodorsal tegmental (LDT) nuclei indirectly influence the activity of the dorsal striatum and nucleus accumbens through their innervation of dopamine and thalamic neurons, which in turn converge at the same striatal levels. Here we show that cholinergic neurons in the brainstem also provide a direct innervation of the striatal complex. By the expression of fluorescent proteins in choline acetyltransferase (ChAT)::Cre(+) transgenic rats, we selectively labeled cholinergic neurons in the rostral PPN, caudal PPN, and LDT. We show that cholinergic neurons topographically innervate wide areas of the striatal complex: rostral PPN preferentially innervates the dorsolateral striatum, and LDT preferentially innervates the medial striatum and nucleus accumbens core in which they principally form asymmetric synapses. Retrograde labeling combined with immunohistochemistry in wild-type rats confirmed the topography and cholinergic nature of the projection. Furthermore, transynaptic gene activation and conventional double retrograde labeling suggest that LDT neurons that innervate the nucleus accumbens also send collaterals to the thalamus and the dopaminergic midbrain, thus providing both direct and indirect projections, to the striatal complex. The differential activity of cholinergic interneurons and cholinergic neurons of the brainstem during reward-related paradigms suggest that the two systems play different but complementary roles in the processing of information in the striatum.

Abnormal functional connectivity between motor cortex and pedunculopontine nucleus following chronic dopamine depletion.

The activity of the basal ganglia is altered in Parkinson's disease (PD) as a consequence of the degeneration of dopamine neurons in the substantia nigra pars compacta. This results in aberrant discharge patterns and expression of exaggerated oscillatory activity across the basal ganglia circuit. Altered activity has also been reported in some of the targets of the basal ganglia, including the pedunculopontine nucleus (PPN), possibly due to its close interconnectivity with most regions of the basal ganglia. However, the nature of the involvement of the PPN in the pathophysiology of PD has not been fully elucidated. Here, we recorded local field potentials in the motor cortex and the PPN in the 6-hydroxydopamine (6-OHDA)-lesioned rat model of PD under urethane anesthesia. By means of linear and nonlinear statistics, we analyzed the synchrony between the motor cortex and the PPN and the delay in the interaction between these two structures. We observed the presence of coherent activity between the cortex and the PPN in low (5-15 Hz)- and high (25-35 Hz)-frequency bands during episodes of cortical activation. In each case, the cortex led the PPN. Dopamine depletion strengthened the interaction of the low-frequency activities by increasing the coherence specifically in the theta and alpha ranges and reduced the delay of the interaction in the gamma band. Our data show that cortical inputs play a determinant role in leading the coherent activity with the PPN and support the involvement of the PPN in the pathophysiology of PD.

Differential modulation of excitatory and inhibitory striatal synaptic transmission by histamine.

Information processing in the striatum is critical for basal ganglia function and strongly influenced by neuromodulators (e.g., dopamine). The striatum also receives modulatory afferents from the histaminergic neurons in the hypothalamus which exhibit a distinct diurnal rhythm with high activity during wakefulness, and little or no activity during sleep. In view of the fact that the striatum also expresses a high density of histamine receptors, we hypothesized that released histamine will affect striatal function. We studied the role of histamine on striatal microcircuit function by performing whole-cell patch-clamp recordings of neurochemically identified striatal neurons combined with electrical and optogenetic stimulation of striatal afferents in mouse brain slices. Bath applied histamine had many effects on striatal microcircuits. Histamine, acting at H(2) receptors, depolarized both the direct and indirect pathway medium spiny projection neurons (MSNs). Excitatory, glutamatergic input to both classes of MSNs from both the cortex and thalamus was negatively modulated by histamine acting at presynaptic H(3) receptors. The dynamics of thalamostriatal, but not corticostriatal, synapses were modulated by histamine leading to a facilitation of thalamic input. Furthermore, local inhibitory input to both classes of MSNs was negatively modulated by histamine. Subsequent dual whole-cell patch-clamp recordings of connected pairs of striatal neurons revealed that only lateral inhibition between MSNs is negatively modulated, whereas feedforward inhibition from fast-spiking GABAergic interneurons onto MSNs is unaffected by histamine. These findings suggest that the diurnal rhythm of histamine release entrains striatal function which, during wakefulness, is dominated by feedforward inhibition and a suppression of excitatory drive.

Functional alterations to the nigrostriatal system in mice lacking all three members of the synuclein family.

The synucleins (α, ß, and γ) are highly homologous proteins thought to play a role in regulating neurotransmission and are found abundantly in presynaptic terminals. To overcome functional overlap between synuclein proteins and to understand their role in presynaptic signaling from mesostriatal dopaminergic neurons, we produced mice lacking all three members of the synuclein family. The effect on the mesostriatal system was assessed in adult (4- to 14-month-old) animals using a combination of behavioral, biochemical, histological, and electrochemical techniques. Adult triple-synuclein-null (TKO) mice displayed no overt phenotype and no change in the number of midbrain dopaminergic neurons. TKO mice were hyperactive in novel environments and exhibited elevated evoked release of dopamine in the striatum detected with fast-scan cyclic voltammetry. Elevated dopamine release was specific to the dorsal not ventral striatum and was accompanied by a decrease of dopamine tissue content. We confirmed a normal synaptic ultrastructure and a normal abundance of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complexes in the dorsal striatum. Treatment of TKO animals with drugs affecting dopamine metabolism revealed normal rate of synthesis, enhanced turnover, and reduced presynaptic striatal dopamine stores. Our data uniquely reveal the importance of the synuclein proteins in regulating neurotransmitter release from specific populations of midbrain dopamine neurons through mechanisms that differ from those reported in other neurons. The finding that the complete loss of synucleins leads to changes in dopamine handling by presynaptic terminals specifically in those regions preferentially vulnerable in Parkinson's disease may ultimately inform on the selectivity of the disease process.

Subpopulations of cholinergic, GABAergic and glutamatergic neurons in the pedunculopontine nucleus contain calcium-binding proteins and are heterogeneously distributed.

Neurons in the pedunculopontine nucleus (PPN) are highly heterogeneous in their discharge properties, their neurochemical markers, their pattern of connectivity and the behavioural processes in which they participate. Three main transmitter phenotypes have been described, cholinergic, GABAergic and glutamatergic, and yet electrophysiological evidence suggests heterogeneity within these subtypes. To gain further insight into the molecular composition of these three populations in the rat, we investigated the pattern of expression of calcium binding proteins (CBPs) across distinct regions of the PPN and in relation to the presence of other neurochemical markers. Calbindin- and calretinin-positive neurons are as abundant as cholinergic neurons, and their expression follows a rostro-caudal gradient, whereas parvalbumin is expressed by a low number of neurons. We observed a high degree of expression of CBPs by GABAergic and glutamatergic neurons, with a large majority of calbindin- and calretinin-positive neurons expressing GAD or VGluT2 mRNA. Notably, CBP-positive neurons expressing GAD mRNA were more concentrated in the rostral PPN, whereas the caudal PPN was characterized by a higher density of CBP-positive neurons expressing VGluT2 mRNA. In contrast to these two large populations, in cholinergic neurons expression of calretinin is observed only in low numbers and expression of calbindin is virtually non-existent. These findings thus identify novel subtypes of cholinergic, GABAergic and glutamatergic neurons based on their expression of CBPs, and further contribute to the notion of the PPN as a highly heterogeneous structure, an attribute that is likely to underlie its functional complexity.

Living on the edge with too many mouths to feed: why dopamine neurons die.

Although genes, protein aggregates, environmental toxins, and other factors associated with Parkinson's disease (PD) are widely distributed in the nervous system and affect many classes of neurons, a consistent feature of PD is the exceptional and selective vulnerability of dopamine (DA) neurons of the SNc. What is it about these neurons, among all other neurons in the brain, that makes them so susceptible in PD? We hypothesize that a major contributory factor is the unique cellular architecture of SNc DA neuron axons. Their large, complex axonal arbour puts them under such a tight energy budget that it makes them particularly susceptible to factors that contribute to cell death, including unique molecular characteristics associated with SNc DA neurons and nonspecific, nervous-system-wide factors.

Cortical and thalamic innervation of direct and indirect pathway medium-sized spiny neurons in mouse striatum.

The striatum receives major excitatory inputs from the cortex and thalamus that predominantly target the spines of medium-sized spiny neurons (MSNs). We aimed to determine whether there is any selectivity of these two excitatory afferents in their innervation of direct and indirect pathway MSNs. To address this, we used bacterial artificial chromosome transgenic mice, in which enhanced green fluorescent protein (EGFP) reports the presence of D(1) or D(2) dopamine receptor subtypes, markers of direct and indirect pathway MSNs, respectively. Excitatory afferents were identified by the selective expression of vesicular glutamate transporter type 1 (VGluT1) by corticostriatal afferents and vesicular glutamate transporter type 2 (VGluT2) by thalamostriatal afferents. A quantitative electron microscopic analysis was performed on striatal tissue from D(1) and D(2) mice that was double immunolabeled to reveal the EGFP and VGluT1 or VGluT2. We found that the proportion of synapses formed by terminals derived from the cortex and thalamus was similar for both direct and indirect pathway MSNs. Furthermore, qualitative analysis revealed that individual cortical or thalamic terminals form synapses with both direct and indirect pathway MSNs. Similarly, we observed a convergence of cortical and thalamic inputs onto individual MSNs of both direct and indirect pathway: individual EGFP-positive structures received input from both VGluT2-positive and VGluT2-negative terminals. These findings demonstrate that direct and indirect pathway MSNs are similarly innervated by cortical and thalamic afferents; both projections are thus likely to be critical in the control of MSNs and hence play fundamental roles in the expression of basal ganglia function.

Dopaminergic axons in different divisions of the adult rat striatal complex do not express vesicular glutamate transporters.

Midbrain dopamine neurons signal rapid information about rewards and reward-related events. It has been suggested that this fast signal may, in fact, be conveyed by co-released glutamate. Evidence that dopamine neurons co-release glutamate comes largely from studies involving cultured neurons or tissue from young animals. Recently, however, it has been shown that this dual glutamatergic/dopaminergic phenotype declines with age, and can be induced by injury, suggesting that it is not a key feature of adult dopamine neurons. Here, we provide further support for this view by showing that dopaminergic axons and terminals in subregions of the adult striatum do not express vesicular glutamate transporters (VGluT1, VGluT2 or VGluT3). Striatal tissue from the adult rat was immunolabelled to reveal tyrosine hydroxylase (TH; biosynthetic enzyme of dopamine) and one of the three known VGluTs. Importantly, we compared the immunogold labelling for each of the VGluTs associated with TH-positive structures with background labelling at the electron microscopic level. In addition, we carried out a subregional analysis of the core and shell of the nucleus accumbens. We found that dopaminergic axons and terminals in the dorsolateral striatum and ventral striatum (nucleus accumbens core and shell) do not express VGluT1, VGluT2 or VGluT3. We conclude, therefore, that in the normal, adult rat striatum, dopaminergic axons do not co-release glutamate.

Activity of neurochemically heterogeneous dopaminergic neurons in the substantia nigra during spontaneous and driven changes in brain state.

Dopaminergic neurons of the substantia nigra (SN) and ventral tegmental area (VTA) are collectively implicated in motor- and reward-related behaviors. However, dopaminergic SN and VTA neurons differ on several functional levels, and dopaminergic SN neurons themselves vary in their intrinsic electrical properties, neurochemical characteristics and connections. This heterogeneity is not only important for normal function; calbindin (CB) expression by some dopaminergic SN neurons has been linked with their increased survival in Parkinson's disease. To test whether the activity of CB-negative and CB-positive dopaminergic SN neurons differs during distinct spontaneous and driven brain states, we recorded single units in anesthetized rats before, during and after aversive somatosensory stimuli. Recorded neurons were juxtacellularly labeled, confirmed to be dopaminergic, and tested for CB immunoreactivity. During cortical slow-wave activity, the firing of most dopaminergic neurons was slow and regular/irregular and unrelated to cortical slow oscillations. During spontaneous cortical activation, dopaminergic SN neurons fired in a more regular manner, with fewer bursts, but did not change their firing rate. Regardless of brain state, CB-negative dopaminergic neurons fired significantly faster than CB-positive dopaminergic neurons. This difference in firing rate was not mirrored by different firing patterns. Most CB-negative and CB-positive dopaminergic neurons did not respond to the aversive stimuli; of those that did respond, most were inhibited. We conclude that CB-negative and CB-positive dopaminergic neurons exhibit different activities in vivo. Furthermore, the firing of dopaminergic SN neurons is brain state-dependent, and, unlike dopaminergic VTA neurons, they are not commonly recruited or inhibited by aversive stimuli.

Short-latency visual input to the subthalamic nucleus is provided by the midbrain superior colliculus.

The subthalamic nucleus (STN) is one of the principal input nuclei of the basal ganglia. Using electrophysiological techniques in anesthetized rats, we show that the STN becomes responsive to visual stimuli at short latencies when local disinhibitory injections are made into the midbrain superior colliculus (SC), an important subcortical visual structure. Significantly, only injections into the lateral, but not medial, deep layers of the SC were effective. Corresponding disinhibition of primary visual cortex also was ineffective. Complementary anatomical analyses revealed a strong, regionally specific projection from the deep layers of the lateral SC to neurons in rostral and dorsal sectors of the STN. Given the retinocentric organization of the SC, these results suggest that lower-field stimuli represented in the lateral colliculus have a direct means of communicating with the basal ganglia via the STN that is not afforded to visual events occurring in the upper visual field.