The precise localization of Dl and D2 dopamine receptors within striatal neurons and circuits is crucial information for further understanding dopamine pharmacology. We have used subtype specific polyclonal and monoclonal antibodies against Dl and D2 dopamine receptors to determine their cellular and subcellular distributions, their colocalization, and their differential connectivity with motor cortical afferents labeled either by lesion-induced degeneration or by anterograde transport of biotinylated dextrans. Dl and D2 are primarily expressed in medium-sized neurons and spiny dendrites. Axon terminals containing Dl were rare whereas DS-immunoreactive axon terminals forming symmetrical synapses with dendrites and spines were common. In 2 p.m sections, Dl was localized to 53% of neurons, and D2 to 48% of neurons, while mixing Dl and D2 antibodies labeled 78%. By electron microscopy, Dl was localized to 43% of dendrites and 38% of spines while D2 was localized to 38% of dendrites and 48% of spines. Combining Dl and D2 antibodies resulted in the labeling of 88.5% of dendrites and 92.6% of spines. Using different chromogens for Dl and D2, colocalization was not observed. lpsilateral motor corticostriatal afferents were primarily axospinous and significantly more synapsed with Dl than DS-positive spines (65% vs 47%). Contralateral motor corticostriatal afferents were frequently axodendritic and no difference in their frequency of synapses with Dl and D2 dendrites and spines was observed. These findings demonstrate differential patterns of expression of Dl and D2 receptorsin striatal neurons and axon terminals and their differential involvement in motor corticostriatal circuits.[

. The regulation of activity in the subthalamic nucleus (STN) by GABAergic inhibition from the reciprocally connected globus pallidus (GP) plays an important role in normal movement and disorders of movement. To determine the precise manner in which GABAergic synaptic input, acting at A-type receptors, influences the firing of STN neurons, we recorded the response of STN neurons to GABA-A inhibitory postsynaptic potentials (IPSPs) that were evoked by supramaximal electrical stimulation of the internal capsule using the perforatedpatch technique in slices at 37°C. The mean equilibrium potential of the GABA-A IPSP (EGABA-A IPSP) was Ϫ79.4 Ϯ 7.0 mV. Single IPSPs disrupted the spontaneous oscillation that underlies rhythmic single-spike firing in STN neurons. As the magnitude of IPSPs increased, the effectiveness of prolonging the interspike interval was related more strongly to the phase of the oscillation at which the IPSP was evoked. Thus the largest IPSPs tended to reset the oscillatory cycle, whereas the smallest IPSPs tended to produce relatively phaseindependent delays in firing. Multiple IPSPs were evoked at various frequencies and over different periods and their impact was studied on STN neurons held at different levels of polarization. Multiple IPSPs reduced and/or prevented action potential generation and/or produced sufficient hyperpolarization to activate a rebound depolarization, which generated a single spike or restored rhythmic spiking and/or generated a burst of activity. The pattern of IPSPs and the level of polarization of STN neurons were critical in determining the nature of the response. The duration of bursts varied from 20 ms to several hundred milliseconds, depending on the intrinsic rebound properties of the postsynaptic neuron. These data demonstrate that inhibitory input from the GP can produce a range of firing patterns in STN neurons, depending on the number and frequencies of IPSPs and the membrane properties and voltage of the postsynaptic neuron.

In order to clarify the origin and to examine the neurochemistry and synaptology of the projection from the mesopontine tegmentum (MTg) to the subthalamic nucleus (STN), rats received discrete deposits of anterograde tracers in different regions of the MTg. Anterogradely labeled fibers were examined in the light and electron microscopes. The distribution of GABA or glutamate immunoreactivity was examined by post- embedding immunocytochemistry. The anterograde tracing demonstrated that the projection to the STN arises from at least three divisions of the MTg: the area defined by the cholinergic neurons of the pedunculopontine region (PPN-Ch 5), the more medial and largely noncholinergic midbrain extrapyramidal area (MEA) and to a lesser extent the laterodorsal tegmental nucleus (LDTg). Post-embedding immunocytochemistry revealed that there are GABA-immunopositive and immunonegative components to this projection and at least a proportion of the GABA-immunonegative component is enriched in glutamate immunoreactivity. The similarity of the morphology, trajectory and synaptology of the anterogradely labeled fibers and the choline acetyltransferase (ChAT)-immunopositive fibers supports the proposal that at least part of the projection is cholinergic. The terminals anterogradely labeled from the MTg and the ChAT-immunoreactive terminals form asymmetrical synapses with the dendrites and spines of subthalamic neurons. Both anterogradely labeled and ChAT-positive terminals make convergent synaptic contacts with GABA-immunoreactive terminals that form symmetrical synaptic contacts and are probably derived from the globus pallidus. Taken together these findings imply that the MTg sends cholinergic, GABAergic and glutamatergic projections to the STN where at least one of the functional roles is to modulate the indirect pathway of information flow through the basal ganglia that is carried via the pallidosubthalamic projection.

Reciprocally connected glutamatergic subthalamic and GABAergic globus pallidus neurons have recently been proposed to act as a generator of low-frequency oscillatory activity in Parkinson's disease. To determine whether GABA(A) receptor-mediated synaptic potentials could theoretically generate rebound burst firing in subthalamic neurons, a feature that is central to the proposed oscillatory mechanism, we determined the equilibrium potential of GABA(A) current (E(GABA(A))) and the degree of hyperpolarization required for rebound firing using perforated-patch recording. In the majority of neurons that fired rebounds, E(GABA(A)) was equal to or more hyperpolarized than the hyperpolarization required for rebound burst firing. These data suggest that synchronous activity of pallidal inputs could underlie rhythmic bursting activity of subthalamic neurons in Parkinson's disease.

In adult rats with a unilateral 6-hydroxydopamine-induced destruction of the nigrostriatal dopamine (DA) pathway, grafts of embryonic substantia nigra can establish a new dopaminergic terminal fiber plexus in the previously denervated neostriatum and compensate for some of the behavioral deficits induced by the nigrostriatal lesion. In the present study the synaptic connections of the ingrowing DA fibers from the graft were analyzed ultrastructurally, using immunocytochemical localization of tyrosine hydroxylase (TH), in animals whose lesion-induced motor asymmetry had been completely compensated for by the nigral grafts. In two of the animals, horseradish peroxidase-wheatgerm agglutinin conjugate was injected into the graft in order to trace possible reciprocal afferent connections to the graft from the host striatum. TH-immunoreactive axons from the graft were seen to make abundant symmetric synapses with neuronal elements in the host neostriatum. Between 85 and 90% of these synapses were on dendritic shafts and spines, and the rest were on neuronal perikarya. Two principal targets were identified: dendrites of spiny neurons, the majority of which are likely to be striatal projection neurons; and the cell bodies of giant neurons, most (or perhaps all) of which are known to be cholinergic interneurons. The synapses made on dendritic spines, which constituted about 40% of all TH-positive synapses formed by the TH-positive neurons in the graft, resembled those seen in normal animals, both in that they made contacts with spine necks and in that they invariably were associated with an asymmetric TH-negative synapse contacting the spine head. The innervation of the giant cell perikarya, which constituted about 6% of all TH-positive synapses found, was strikingly abnormal in that the graft-derived TH-positive fibers formed dense pericellular "baskets" selectively around the giant cell bodies. Such arrangements were never seen in the normal striatum, nor did they occur in the intact contralateral striatum in the grafted animals. It is proposed that this apparent dopaminergic hyperinnervation from the graft could provide a powerful inhibition of the cholinergic interneurons in the reinnervated host striatum, and that such an inhibitory mechanism could assist in the graft-induced functional recovery by potentiating the functional effects of DA synapses terminating on the spiny efferent neurons. This dual innervation may thus help to explain why restoration of only a small proportion of the striatal DA innervation by the graft is sufficient to induce complete compensation of, e.g., motor asymmetry in the lesioned rats.(ABSTRACT TRUNCATED AT 400 WORDS)

To determine the principles of synaptic innervation of neurons in the entopeduncular nucleus and subthalamic nucleus by neurons of functionally distinct regions of the pallidal complex, double anterograde labeling was carried out at both light and electron microscopic levels in the rat. Deposits of the anterograde tracers Phaseolus vulgaris-leucoagglutinin and biotinylated dextran amine were placed in different functional domains of the pallidal complex in the same animals. The tracer deposits in the ventral pallidum and the globus pallidus gave rise to GABA-immunopositive projections to the entopeduncular nucleus, the subthalamic nucleus, and the more medial lateral hypothalamus that were largely segregated but overlapped at the interface between the two fields of projection. In these regions the proximal parts of individual neurons in the entopeduncular nucleus, lateral hypothalamus, and subthalamic nucleus received synaptic input from terminals derived from both the ventral pallidum and the globus pallidus. Furthermore, the analysis of the afferent synaptic input to the dendrites of neurons in the subthalamic nucleus that cross functional boundaries of the nucleus defined by the pallidal inputs, revealed that terminals with the morphological and neurochemical characteristics of those derived from the pallidal complex make synaptic contact with all parts of the dendritic tree, including distal regions. It is concluded that functionally diverse information carried by the descending projections of the pallidal complex is synaptically integrated by neurons of the entopeduncular nucleus, lateral hypothalamus, and subthalamic nucleus by two mechanisms. First, neurons located at the interface between functionally distinct, but topographically adjacent, projections could integrate diverse information by means of the synaptic convergence at the level of the cell body and proximal dendrites. Second, because the distal dendrites of neurons in the subthalamic nucleus receive input from the pallidum, those that extend across two distinct domains of pallidal input could also provide the morphological basis of integration.

The inhibitory amino acid ␥-aminobutyric acid (GABA) is widely distributed in the basal ganglia. It plays a critical role in the functioning of the striatum as it is the transmitter of projection neurons and sub-populations of interneurons, as well as afferents from the globus pallidus. Some of the factors controlling GABA transmission are the type(s) of GABA receptor expressed at the site of transmission, their subunit composition, and their location in relation to GABA release sites. To address these issues, we examined the sub-cellular localization of subunits of the GABA A receptor in the striatum of the rat. Sections of freeze-substituted, Lowicryl-embedded striatum were immunolabelled by the post-embedding immunogold technique with antibodies specific for subunits of the GABA A receptor. Immunolabelling for ␣1, ␤2/3, and ␥2 GABA A receptor subunits was primarily located at symmetrical synapses on perikarya, dendrites, and spines. Quantitative analysis of the distribution of immunolabelling for the ␤2/3 subunits revealed that the majority of membrane associated immunogold particles were at synapses and that, on average for the whole population, they were evenly distributed across the synapse. Double labelling for the ␤2/3 subunits and for GABA itself revealed that receptor-positive synapses were formed by at least two populations of terminals. One population (59.3%) of terminals forming receptor-positive synapses was positive for GABA, whereas the other (40.7%) had low or undetectable levels of GABA. Furthermore, the post-synaptic neurons were characterised on neurochemical and morphological grounds as both medium spiny neurons and GABA interneurons. Triple immunolabelling revealed the co-localization of ␣1, ␤2/3, and ␥2 subunits at some symmetrical axodendritic synapse. It is concluded that fast GABA A -mediated transmission occurs primarily at symmetrical synapses within the striatum, that the populations of boutons giving rise to receptor-positive synapses are heterogeneous, and that previously reported co-existence of different subunits of the GABA A receptor at the cellular level also occurs at the level of individual synapses.

The axon initial segment (AIS) is the site of initiation of action potentials and influences action potential waveform, firing pattern, and rate. In view of the fundamental aspects of motor function and behavior that depend on the firing of substantia nigra pars compacta (SNc) dopaminergic neurons, we identified and characterized their AIS in the mouse. Immunostaining for tyrosine hydroxylase (TH), sodium channels (Na ) and ankyrin-G (Ank-G) was used to visualize the AIS of dopaminergic neurons. Reconstructions of sampled AIS of dopaminergic neurons revealed variable lengths (12-60 μm) and diameters (0.2-0.8 μm), and an average of 50% reduction in diameter between their widest and thinnest parts. Ultrastructural analysis revealed submembranous localization of Ank-G at nodes of Ranvier and AIS. Serial ultrathin section analysis and 3D reconstructions revealed that Ank-G colocalized with TH only at the AIS. Few cases of synaptic innervation of the AIS of dopaminergic neurons were observed. mRNA in situ hybridization of brain-specific Na subunits revealed the expression of Na 1.2 by most SNc neurons and a small proportion expressing Na 1.6. The presence of sodium channels, along with the submembranous location of Ank-G is consistent with the role of AIS in action potential generation. Differences in the size of the AIS likely underlie differences in firing pattern, while the tapering diameter of AIS may define a trigger zone for action potentials. Finally, the conspicuous expression of Na 1.2 by the majority of dopaminergic neurons may explain their high threshold for firing and their low discharge rate.