Interconnected parallel circuits between rat nucleus accumbens and thalamus revealed by retrograde transynaptic transport of pseudorabies virus

Abstract

One of the primary outputs of the nucleus accumbens is directed to the mediodorsal thalamic nucleus (MD) via its projections to the ventral pallidum (VP), with the core and shell regions of the accumbens projecting to the lateral and medial aspects of the VP, respectively. In this study, the multisynaptic organization of nucleus accumbens projections was assessed using intracerebral injections of an attenuated strain of pseudorabies virus, a neurotropic alpha herpesvirus that replicates in synaptically linked neurons. Injection of pseudorabies virus into different regions of the MD or reticular thalamic nucleus (RTN) produced retrograde transynaptic infections that revealed multisynaptic interactions between these areas and the basal forebrain. Immunohistochemical localization of viral antigen at short postinoculation intervals confirmed that the medial MD (m-MD) receives direct projections from the medial VP, rostral RTN, and other regions previously shown to project to this region of the thalamus. At longer survival intervals, injections confined to the m-MD resulted in transynaptic infection of neurons in the accumbens shell but not in the core. Injections that also included the central segment of the MD produced retrograde infection of neurons in the lateral VP and the polymorph (pallidal) region of the olfactory tubercle (OT) and transynaptic infection of a small number of neurons in the rostral accumbens core. Injections in the lateral MD resulted in retrograde infection in the globus pallidus (GP) and in transynaptic infection in the caudate-putamen. Viral injections into the rostroventral pole of the RTN infected neurons in the medial and lateral VP and at longer postinoculation intervals, led to transynaptic infection of scattered neurons in the shell and core. Injection of virus into the intermediate RTN resulted in infection of medial VP neurons and second-order infection of neurons in the accumbens shell. Injections in the caudal RTN or the lateral MD resulted in direct retrograde labeling of cells within the GP and transynaptic infection of neurons in the caudate-putamen. These results indicate that the main output of VP neurons receiving inputs from the shell of the accumbens is heavily directed to the m-MD, whereas a small number of core neurons appear to influence the central MD via the lateral VP. Further segregation in the flow of information to the MD is apparent in the organization of VP and GP projections to subdivisions of the RTN that give rise to MD afferents. Collectively, these data provide a morphological basis for the control of the thalamocortical system by ventral striatal regions, in which parallel connections to the RTN may exert control over activity states of cortical regions.

Fig. 1.

The diffusion of CT and PRV from a common site of injection is illustrated in this figure. The two tracers were mixed in equal parts, and 200 nl was injected into the mediodorsal nucleus (MDN) at 10 nl/min. A–F illustrate localization of PRV (A, C,E) or CT (B, D,F) in adjacent sections through rostral (A, B), intermediate (C,D), and caudal (E,F) levels of the MDN. PRV immunoreactivity is largely confined to the medial region of the MDN in the rostral half of the nucleus, whereas CT exhibits a larger sphere of diffusion throughout the rostrocaudal extent of the MDN. Scale bar (shown inF): 200 μm; all figures are of the same magnification.

Fig. 3.

Injection of PRV into the medial segment of the MD nucleus revealed a disynaptic connection with the basal forebrain.A and B demonstrate the restricted injection site resulting from injection of 100 nl of virus at 10 nl per minute. Neurons displaying PRV immunoreactivity are confined to the medial segment of the nucleus with no apparent spread to the central segment or adjacent thalamic nuclei. At short postinoculation intervals, infected neurons were present in the medial/rostral aspect of the VP (C, open block arrow,inset) but were not found in either division of the nucleus accumbens. The inset in Cillustrates the region that is shown at higher magnification inD, in which calbindin immunoreactivity defines the limits of the core (NAco) and shell (NAsh) regions of the nucleus accumbens. With more advanced infection, more infected neurons were apparent in the medial VP, and they exhibited neuropathological changes characteristic of advanced viral replication (E, F). In addition, the virus passed transynaptically to infect neurons in the shell, but not in the core, of the nucleus accumbens (E,G). See text for additional details of the experiments and controls that established the route of viral transport through this circuitry. The area marked by the open and closed block arrows in E are shown at higher magnification in F and G, respectively. Scale bars: A, C, E, 200 μm; B, D, 100 μm; F,G, inset in C, 50 μm.

Fig. 5.

Injections of PRV that involved the c-MD and l-MD (A) produced a pattern of infection that differed from that produced by injection of virus into the m-MD. Infected first-order neurons were present in the intermediate and caudal RTN (B), the GP, (C), and the lateral VP (E, F). At longer postinoculation intervals, we did not observe transynaptic infection of neurons in the shell of the nucleus accumbens (D), but did observe occasional neurons in the core at rostral levels (G). The cells marked by the open block arrow inE are shown at higher magnification in F. The prominent viral immunoreactivity in the somatodendritic compartments of neurons in the GP and VP (C,E) and pathological changes in some of the infected cells (F, arrow) indicate that these cells are in an advanced stage of infection. Hb, Habenula; GP, globus pallidus; St, striatum; AC, anterior commissure. Scale bars:A–D (shown in C), 200 μm; E, 100 μm; F, G(shown in G), 20 μm.

Fig. 6.

Injection of PRV into the rostral and ventral portion of the RTN (A) labeled elements of the MD and basal forebrain circuitry revealed by injection of virus into the l-MD (compare with Fig. 5). The center of the injection site is marked by the asterisk in A. Retrogradely infected first-order neurons were observed in the lateral portion of the VP (B) as well as in the c-MD and l-MD (C). The morphology of the neurons marked by the large andsmall open block arrows in C are shown at higher magnification in D and E, respectively. Transynaptic infection of small numbers of neurons was apparent in the medial VP and nucleus accumbens shell (F) as well as in the rostral portion of the core (G). Scale bars: A, C,F, 200 μm; B, 100 μm;D, E, 20 μm.

Fig. 7.

Injection of virus into the caudal RTN produced a pattern of infection distinct from that produced by injection of the rostral RTN. The site of injection is illustrated in Aand B, with the asterisks marking the position of the tip of the injection cannula. Scattered infected first-order neurons were present in the l-MD (C); the neuron marked by the open block arrow inC is shown at higher magnification in theinset. Larger numbers of first-order neurons were present in the GP (D, E). At longer survival times, striatal medium spiny neurons were heavily infected (G, bottom of field), and we also noted viral immunoreactivity in large spiny interneurons of the striatum that exhibited a morphology similar to that of cholinergic interneurons (F, open block arrow). The appearance of viral immunoreactivity in these cells at long survival intervals is consistent with them becoming infected by retrograde transynaptic passage of virus from the medium spiny neurons. No infected neurons were observed in either the core or the shell of the nucleus accumbens in these animals. Scale bars: A–C, 200 μm; D, 50 μm; E, F,inset in C, 20 μm.

Fig. 2.

Examination of the cingulate cortex after injection of PRV into the lateral subdivision of the MDN demonstrates that this strain of virus is only transported retrogradely from the site of intracerebral injection. In A, the most prominent viral immunoreactivity is present in neurons found in deep layers of the cortex. Densely staining somata and dendrites of these neurons reveal the clear morphological features of pyramidal neurons and also demonstrates that these neurons are in an advanced stage of infection. In contrast, only scattered neurons in an early stage of infection are apparent in superficial layers. In many of these cells, viral immunoreactivity is confined to the cell nuclei (B, small arrows), and when it is apparent in the cytoplasm (open block arrow), the staining reveals that the cells are small interneurons rather than projection neurons. These data indicate that retrograde transport of virus from the MDN produced the first-order infection of pyramidal neurons in deeper layers of cingulate cortex and that transynaptic passage of virus from these cells produced the temporally delayed infection of interneurons in superficial layers. Scale bars:A, 100 μm; B, 50 μm.

Fig. 4.

Injection of the m-MD also revealed a topographically organized afferent input from neurons in the RTN. After restricted injections of this subdivision, infected neurons were present in the ventromedial portion of the rostral RTN (B), but were not present in the caudal RTN (A, arrowheads). Scale bars:A, 200 mm; B, 100 mm.

Fig. 10.

Structures infected after a viral injection restricted to the l-MD. The injection site of this representative case is shown as a hatched area; solid circlesrepresent the distribution of first-order projection neurons;open circles show the location of second-order projection neurons.

Fig. 8.

PRV injection in the m-MD resulted in a consistent pattern of infection across structures. Symbolsrepresent infected neurons and their placement in 12 representative drawings of coronal sections of the rat brain, modified from a stereotaxic rat brain atlas (Paxinos and Watson, 1986). The injection sites of two cases are represented with hatched areas, and the results from each animal are shown with different symbols (circles, data from injection shown as vertical hatch; triangles, data from injection shown ashorizontal hatch). Solid symbolsrepresent the location of first-order projection neurons, andopen symbols show the distribution of second-order projection neurons.

Fig. 9.

Injection of virus that included the c-MD exhibited infection of some common structures. Symbolsrepresent the distribution of infected neurons after injection of PRV in selected cases. Circles show the distribution of infection after an injection including both the m-MD and the c-MD (injection site shown with vertical hatch).Triangles represent the distribution of infected neurons after an injection that included both the c-MD and the l-MD (injection site represented with horizontal hatch). The distribution of infection in structures labeled in both cases is shown in bold; the distribution of infection observed in any of the cases but not on the other is shown ingray.

Fig. 11.

Injections of virus in the rostral RTN resulted in a specific pattern of infection. Circles andtriangles represent first-order infected neurons (solid) and second-order infected neurons (open) for two selected cases. The distribution of infected neurons after the small injection (gray) is shown with triangles, whereas the location of infected cells after the larger injection (hatched) is shown with circles.

Fig. 12.

Co-injection of PRV and CT in the intermediate RTN results in similar first-order projections. Circlesrepresent PRV-infected neurons (solid, first order;open, second order). Small × symbols represent the distribution of CT-labeled neurons. The extension of PRV infection in the injection site is shown as a dark area, and the larger extension of CT around the injection site is shown as ahatched area.

Fig. 13.

Distribution of infected neurons in a case representative of injections in the caudal RTN. The injection site is shown as a hatched area; solid trianglesrepresent the distribution of first-order projection neurons, andopen triangles show the distribution of second-order projection neurons.

Fig. 14.

Parallel circuits linking the CPu, accumbens core, and accumbens shell with pallidal and thalamocortical systems. The arrows summarize the most consistent data obtained after PRV injections into the MD or RTN regions. Green arrows represent circuitry involving the accumbens shell and medial VP, red arrows show circuits involving the accumbens core and lateral VP/pallidal OT, and blue arrows represent circuits originated from the CPu and GP.Gray arrows show cortico-thalamic and cortico-striatal projections not assessed in this study. The m-MD receives very heavy projections via the shell/medial VP-axis. The c-MD receives much weaker projections from the core/lateral VP system and through the polymorph layer of the OT. The l-MD receives projections from the dorsal striatum–GP. In addition, the three striatal regions also project (via VP and GP) to relatively segregated regions within the RTN. In turn, these RTN areas project to different MD segments. Also, the three MD segments receive relatively segregated inputs from prefrontal cortical areas. Overall, these data provide strong evidence for the presence of parallel circuits within the basal ganglia-thalamic projections, which are not completely closed, allowing for interactions among them.