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.

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.

Convergence of cortical and thalamic input to direct and indirect pathway medium spiny neurons in the striatum.

The major afferent innervation of the basal ganglia is derived from the cortex and the thalamus. These excitatory inputs mainly target the striatum where they innervate the principal type of striatal neuron, the medium-sized spiny neurons (MSNs), and are critical in the expression of basal ganglia function. The aim of this work was to test directly whether corticostriatal and thalamostriatal terminals make convergent synaptic contact with individual direct and indirect pathway MSNs. Individual MSNs were recorded in vivo and labelled by the juxtacellular method in the striatum of BAC transgenic mice in which green fluorescent protein reports the expression of dopamine D1 or D2 receptors. After recovery of the neurons, the tissue was immunolabelled for vesicular glutamate transporters type 1 and 2, as markers of cortical and thalamic terminals, respectively. Three of each class of MSNs were reconstructed in 3D and second-order dendrites selected for electron microscopic analysis. Our findings show that direct and indirect pathway MSNs, located in the matrix compartment of the striatum, receive convergent input from cortex and thalamus preferentially on their spines. There were no differences in the pattern of innervation of direct and indirect pathway MSNs, but the cortical input is more prominent in both and synaptic density is greater for direct pathway neurons. The 3D reconstructions revealed no morphological differences between direct and indirect MSNs. Overall, our findings demonstrate that direct and indirect pathway MSNs located in the matrix receive convergent cortical and thalamic input and suggest that both cortical and thalamic inputs are involved in the activation of MSNs.

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.

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.

A dopaminergic axon lattice in the striatum and its relationship with cortical and thalamic terminals.

Interactions between glutamatergic corticostriatal afferents and dopaminergic nigrostriatal afferents are central to basal ganglia function. The thalamostriatal projection provides a glutamatergic innervation of similar magnitude to the corticostriatal projection. We tested the hypotheses that (1) thalamostriatal synapses have similar spatial relationships with dopaminergic axons as corticostriatal synapses do and (2) the spatial relationships between excitatory synapses and dopaminergic axons are selective associations. We examined at the electron microscopic level rat striatum immunolabeled to reveal vesicular glutamate transporters (VGluTs) 1 and 2, markers of corticostriatal and thalamostriatal terminals, respectively, together with tyrosine hydroxylase (TH) to reveal dopaminergic axons. Over 80% of VGluT-positive synapses were within 1 microm of a TH-positive axon and >40% were within 1 microm of a TH-positive synapse. Of structures postsynaptic to VGluT1- or VGluT2-positive terminals, 21 and 27%, respectively, were apposed by a TH-positive axon and about half of these made synaptic contact. When structures postsynaptic to VGluT-positive terminals and VGluT-positive terminals themselves were normalized for length of plasma membrane, the probability of them being apposed by, or in synaptic contact with, a TH-positive axon was similar to that of randomly selected structures. Extrapolation of the experimental data to more closely reflect the distribution in 3D reveals that all structures in the striatum are within approximately 1 microm of a TH-positive synapse. We conclude that (1) thalamostriatal synapses are in a position to be influenced by released dopamine to a similar degree as corticostriatal synapses are and (2) these associations arise from a nonselective dopaminergic axon lattice.

Functional diversity and specificity of neostriatal interneurons.

The firing of neostriatal spiny neurons in response to an excitatory input is modulated and sculpted by a variety of factors. Neostriatal interneurons are phenotypically diverse and have properties that enable them to specifically, but differentially, influence the activity of spiny neurons. Each of the three types of GABAergic interneurons produces a strong inhibitory postsynaptic potential in spiny neurons, the function of which is probably to influence the precise timing of action potential firing in either individual or ensembles of spiny neurons. By contrast, the role of cholinergic interneurons is to modulate the sub- and supra-threshold responses of spiny neurons to cortical and/or thalamic excitation, particularly in reward-related activities. Both classes of interneurons are important sites of action of neuromodulators in neostriatum, and act in different but complementary ways to modify the activity of the spiny projection neurons.

Synaptic convergence of motor and somatosensory cortical afferents onto GABAergic interneurons in the rat striatum.

Cortical afferents to the basal ganglia, and in particular the corticostriatal projections, are critical in the expression of basal ganglia function in health and disease. The corticostriatal projections are topographically organized but also partially overlap and interdigitate. To determine whether projections from distinct cortical areas converge at the level of single interneurons in the striatum, double anterograde labeling from the primary motor (M1) and primary somatosensory (S1) cortices in the rat, was combined with immunolabeling for parvalbumin (PV), to identify one population of striatal GABAergic interneurons. Cortical afferents from M1 and S1 gave rise to distinct, but partially overlapping, arbors of varicose axons in the striatum. PV-positive neurons were often apposed by cortical terminals and, in many instances, apposed by terminals from both cortical areas. Frequently, individual cortical axons formed multiple varicosities apposed to the same PV-positive neuron. Electron microscopy confirmed that the cortical terminals formed asymmetric synapses with the dendrites and perikarya of PV-positive neurons as well as unlabelled dendritic spines. Correlated light and electron microscopy revealed that individual PV-positive neurons received synaptic input from axon terminals derived from both motor and somatosensory cortices. These results demonstrate that, within areas of overlap of functionally distinct projections, there is synaptic convergence at the single cell level. Sensorimotor integration in the basal ganglia is thus likely to be mediated, at least in part, by striatal GABAergic interneurons. Furthermore, our findings suggest that the pattern of innervation of GABAergic interneurons by cortical afferents is different from the cortical innervation of spiny projection neurons.