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Although the retrosplenial granular b cortex (Rgb) is situated in a critical position between the hippocampal formation and the neocortex, surprisingly few studies have examined its connections carefully. The present experiments use both anterograde and retrograde tracing techniques to characterize the connections of Rgb. The main cortical projections from Rgb are to the caudal part of the anterior cingulate cortex, area 18b, retrosplenial granular a cortex (Rga), and postsubiculum, and less dense terminal fields are present in the prelimbic and caudal occipital cortices. The major subcortical projections are to the anterior thalamic nuclei and the rostral pontine nuclei, and very small terminal fields are present in the caudal dorsomedial part of the striatum, the reuniens and reticular nuclei of the thalamus, and the mammillary bodies. Contralaterally, Rgb primarily projects to itself, i.e., homotypically, and more sparsely projects to Rga and postsubiculum. In general, the axons from Rgb terminate ipsilaterally in cortical layers I and III-V and contralaterally in layer V, with a smaller number of terminals in layers I and VI. Thalamic projections from Rgb target the anteroventral and laterodorsal nuclei of the thalamus, with only a few axons terminating in the anterodorsal nucleus, the reticular nucleus, and the nucleus reuniens of the thalamus. Rgb is innervated by the anterior cingulate cortex, precentral agranular cortex, cortical area 18b, dorsal subiculum, and postsubiculum. Subcortical projections to Rgb originate mainly in the claustrum, the horizontal limb of the diagonal band of Broca, and the anterior thalamic nuclei. These data demonstrate that, in the rat, Rgb is a major nodal point for the integration and subsequent distribution of information to and from the hippocampal formation, the midline limbic and visual cortices, and the thalamus. Thus, similarly to the entorhinal cortex, Rgb in the rat is a prominent gateway for information exchange between the hippocampal formation and other limbic areas of the brain., (Copyright 2003 Wiley-Liss, Inc.)

The present study characterized the projections of the anterodorsal (AD) and the anteroventral (AV) thalamic nuclei to the limbic cortex. Both AD and AV project to the full extent of the retrosplenial granular cortex in a topographic pattern. Neurons in caudal parts of both nuclei project to rostral retrosplenial cortex, and neurons in rostral parts of both nuclei project to caudal retrosplenial cortex. Within AV, the magnocellular neurons project primarily to the retrosplenial granular a cortex, whereas the parvicellular neurons project mainly to the retrosplenial granular b cortex. AD projections to retrosplenial cortex terminate in very different patterns than do AV projections: The AD projection terminates with equal density in layers I, III, and IV of the retrosplenial granular cortex, whereas, in contrast, the AV projections terminate very densely in layer Ia and less densely in layer IV. Further, both AD and AV project densely to the postsubicular, presubicular, and parasubicular cortices and lightly to the entorhinal (only the most caudal part) cortex and to the subiculum proper (only the most septal part). Rostral parts of AD project equally to all three subicular cortices, whereas neurons in caudal AD project primarily to the postsubicular cortex. Compared to AD, neurons in AV have a less extensive projection to the subicular cortex, and this projection terminates primarily in the postsubicular and presubicular cortices. Further, the AD projection terminates in layers I, II/III, and V of postsubiculum, whereas the AV projection terminates only in layers I and V.

The laterodorsal nucleus (LD) of the thalamus is an important source of thalamic afferents to the limbic cortex, but the topography and lamination of these projections has not been investigated in detail. Using the anterograde transport of Phaseolus vulgaris leucoagglutinin and Fluoro-Ruby, the present study demonstrates that in the rat, LD projects to infraradiata, precentral agranular, retrosplenial, visual (area 18b), subicular, and entorhinal cortices. Each subregion of LD has a distinct pattern of terminals within these cortical areas. The rostral part and the dorsalmost part of LD project densely to retrosplenial granular a (Rga) cortex, presubiculum and parasubiculum. Slightly more caudal parts of dorsal LD project primarily to the postsubiculum. More ventral parts of LD project primarily to retrosplenial dysgranular (Rdg) and retrosplenial granular b (Rgb) cortices. The projection of LD to area 18b originates from cells in the caudalmost part of LD. In each cortical region, LD terminals display distinct laminar patterns. In area 18b and the adjacent Rdg cortex, the LD terminal field is in layers I, III, and IV, but in both the Rgb and Rga cortices the terminal field is located predominantly in layer I. In the postsubiculum the LD terminals are distributed to layers I and III/IV and extend into superficial layer V; in the presubiculum and the parasubiculum the LD terminals are only in the deep layers (i.e., layers IV-VI). A small number of LD axons terminate in the deep layers (i.e., layers IV-VI) of the medial entorhinal cortex. These results indicate that each area of LD has a distinct projection to limbic and adjacent neocortex.

Although the retrosplenial dysgranular cortex (Rdg) is situated both physically and connectionally between the hippocampal formation and the neocortex, few studies have focused on the connections of Rdg. The present study employs retrograde and anterograde anatomical tracing methods to delineate the connections of Rdg. Each projection to Rdg terminates in distinct layers of the cortex. The thalamic projections to Rdg originate in the anterior (primarily the anteromedial), lateral (primarily the laterodorsal), and reuniens nuclei. Those from the anteromedial nucleus terminate predominantely in layers I and IV-VI, whereas the axons arising from the laterodorsal nucleus have a dense terminal plexus in layers I and III-IV. The cortical projections to Rdg originate primarily in the infraradiata, retrosplenial, postsubicular, and areas 17 and 18b cortices. The projections arising from visual areas 18b and 17 predominantly terminate in layer I of Rdg, axons from contralateral Rdg form a dense terminal plexus in layers I-IV, with a smaller number of terminals in layers V and VI, afferents from postsubiculum terminate in layers I and III-V, and the projection from infraradiata cortex terminates in layers I and V-VI. The efferent projections from Rdg are widespread. The major cortical projections from Rdg are to infraradiata, retrosplenial granular, area 18b, and postsubicular cortices. Subcortical projections from Rdg terminate primarily in the ipsilateral caudate and lateral thalamic nuclei and bilaterally in the anterior thalamic nuclei. The efferent projections from Rdg are topographically organized. Rostral Rdg projects to the dorsal infraradiata cortex and the rostral postsubiculum, while caudal Rdg axons terminate predominantely in the ventral infraradiata and the caudal postsubicular cortices. Caudal but not rostral Rdg projects to areas 17 and 18b of the cortex. The Rdg projections to the lateral and anterior nuclei also are organized along the rostral-caudal axis. Together, these data suggest that Rdg integrates thalamic, hippocampal, and neocortical information.

Hippocampal area CA1 provides the major cortical output of the hippocampus, but only its projections to the subiculum and lateral septal nucleus are well characterized. The present study reexamines these extrinsic projections by using anterograde and retrograde tracing techniques. Injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) in the septal one-third of CA1 label axons and terminals in subicular, postsubicular, retrosplenial, perirhinal, and entorhinal cortices, lateral septal nucleus, and diagonal band of Broca. The septal CA1 injections also label terminal fields in contralateral CA1, and in contralateral subicular, postsubicular, perirhinal, and entorhinal cortices. Injections into the splenial one-third of CA1 label axons and terminals in subiculum, postsubiculum, ventral area infraradiata, and lateral septal nucleus, but they do not label axons and terminals on the contralateral side of the brain. Injections in the temporal one-third of CA1 label axons and terminals in subicular, parasubicular, entorhinal, and infraradiata cortices, anterior olfactory nucleus, olfactory bulb, lateral septal nucleus, nucleus accumbens, amygdala, and hypothalamus. The temporal CA1 injections label no axons on the contralateral side of the brain. These data demonstrate that CA1 has more widespread projections than previously appreciated, and they provide the first clear evidence that CA1 projects to the contralateral cortex and to the ipsilateral olfactory bulb, amygdala, and hypothalamus. The results also demonstrate a heterogeneity in the efferent projections originating in different septotemporal levels of CA1.

Although the retrosplenial granular a cortex (Rga) is situated in a critical position between the hippocampal formation and the neocortex, few studies have examined its connections. The present experiments use both retrograde and anterograde tracing techniques to characterize the afferent and efferent connections of Rga. Cortical projections to Rga originate in the ipsilateral area infraradiata, the retrosplenial agranular and granular b cortices, the ventral subiculum, and the contralateral Rga. Subcortical projections originate in the claustrum, the diagonal band of Broca, the thalamus, the midbrain raphe nuclei, and the locus coeruleus. The thalamic projections to Rga originate mainly in the anterodorsal (AD) and laterodorsal (LD) nuclei with sparse projections arising in the anteroventral (AV) and reuniens nuclei. Each projection to Rga terminates in distinct layers of the cortex. The thalamic projection from AD terminates primarily in layers I, III, and IV of Rga, whereas the axons arising from the LD nucleus have a dense terminal plexus only in layer 1. The projections arising from the subiculum end predominantly in layer II, whereas the postsubiculum projects to layers I and III-V. Axons from the contralateral Rga form a dense terminal plexus in layers IV and V, with a smaller number of terminals in layers I and VI. Rga projects ipsilaterally to the AV and LD nuclei of the thalamus and to the anterior cingulate, retrosplenial agranular,a and postsubicular cortices. Contralaterally it projects to the retrosplenial agranular and Rga cortices. Rga projections to the thalamus terminate ipsilaterally in the dorsal part of LD and bilaterally in AV. Together, these data suggest that Rga integrates thalamic with limbic information.

The extrinsic projections to and from the retrosplenial cortex have been studied in detail, but the intrinsic circuitry within this region has been characterized less completely. To further define the internal connections, small injections of the retrograde, fluorescent tracer Fluorogold were made into the retrosplenial cortex of the rat. These injections label neurons in layers II-V of the contralateral homotopic cortex. In layers III-V, the labeled neurons are present over an area much larger than the injection site, but in layer II neurons are labeled in a very precise homotopic pattern. Following these injections, only the neurons in layer II display heavily labeled apical dendrites, and these labeled dendrites form tight bundles in layer Ic and Ib of the cortex and spread out in layer Ia. An examination of Golgi-stained material demonstrates that most of the neurons in layer II are small pyramidal cells with 2-3 small basal dendrites and a single, large apical dendrite that arborizes extensively in layer Ia. To verify the structure of the layer II neurons, they were intracellularly filled with Lucifer yellow. Examination of these labeled cells confirms the observations from the Golgi-stained material and demonstrates that many apical dendrites of the layer II cells angle acutely, apparently to join a bundle and/or avoid an interbundle space. Tract tracing experiments demonstrate that the anteroventral nucleus of the thalamus appears to project selectively to the region containing the dendritic bundles, whereas intracortical projections appear to terminate in layers Ib and Ic in the 30-200 microns spaces between the bundles. Furthermore, the areas containing the bundles display dense AChE staining, but the interbundle spaces are almost free of AChE staining. These findings demonstrate a form of dendritic bundling that is input and output specific and may play an important role in the regulation of thalamic inputs to the cingulate cortex.

The tritiated amino acid autoradiographic method was employed to characterize the patterns of commissural projections originating in the hippocampus of the rat, guinea pig, rabbit, and cat. The results demonstrate that significant differences between species are present despite the overall similarity of the projections. In the rat and cat the commissural connections are widely distributed along the septotemporal axis of the hippocampus, but in the guinea pig and rabbit they are less widely distributed along this axis. Second, within the hippocampus proper the radial distribution of the commissural projection is species specific. In the rat, CA3 commissural projections are present in both the strata oriens and radiatum, but the densest projection is to the stratum oriens. In the guinea pig the radial distribution of this projection is similar to that observed in the rat, but in the rabbit the projection is almost entirely confined to the stratum oriens. In contrast, in the cat the CA3 commissural projection is very dense to the stratum radiatum and sparse to stratum oriens. An analysis of the relative density of label in the molecular layer of the fascia dentata suggests that the density of the commissural projection from CA4 is much greater in the rat and cat than in the guinea pig or rabbit. These results indicate that care must be exercised in the generalization of connectional data between species. The results also suggest a possible explanation for differences observed in the electrophysiology of these connections between species.

The projection of the entorhinal cortex (EA) to the hippocampus in the cat has been studied by electrophysiological methods. Field potentials elicited by EA stimulation sites were measured in the hippocampus (fascia dentata). Different topographic distributions of the amplitude and of the onset latency of average evoked potentials (AEPs) were obtained depending on the place of the stimulation along a lateromedial axis in the Ea. The lateral EA elicited the largest AEPs in the septal part of the hippocampus and the medial EA evoked maximal responses in the temporal part of the hippocampus, while the intermediate part of the EA evoked the largest AEPs in the splenial (intermediate) part of the hippocampus. Unit activity elicited by hippocampal stimulation was measured in the EA. Analysis of the antidromic unit activity showed that the pathways analysed were monosynaptic. Different conduction velocities to the septal part of the hippocampus were found; the pathway from the lateral EA was the fastest and the pathway from the medial EA the slowest. Assuming that the sites of maximal AEP amplitude correspond to the location of the major synaptic inputs, it can be concluded that the active synaptic inputs arising along a latero-medial axis in the EA are distributed within the hippocampus according to a septotemporal axis, although with some overlap between the different projections. Therefore it may be concluded that the hippocampus is not homogeneous with respect to the inputs from the EA. The present observations are discussed regarding anatomical data and putative functional differences between septal and temporal hippocampus.