Neural assemblies and laminar interactions in the cerebral cortex

 Neural assemblies are assumed to become organized and to operate within the cerebral cortex, and so must be constrained by the cytological and physiological properties of this laminated structure. A hypothesis of such assemblies is presented, based on important details of neuronal architecture and physiology in different cortical laminae. Laminae II, III and VI, which are the origin and termination of most cortico-cortical projections, are regarded as the site of storage of most of the information encoded by assemblies – a neuronal ‘library’. Laminae II and III are the most sensitive coincidence detectors, and therefore probably initiate the process of assembly formation. However, these three laminae have very low levels of spontaneous activity in the waking state, and so active cell assemblies cannot base their functioning on these laminae alone. Lamina V pyramidal cells have a much higher level of spontaneous activity. Thus, indirect pathways between ‘library’ cells, via lamina V pyramidal cells, are likely to be more secure than direct ones. It is proposed that direct links between ‘library’ cells become stabilized by Hebbian strengthening, once the recipient ‘library’ cell has been ‘primed’ by neural activity transmitted indirectly via lamina V neurones. Thus lamina V neurones could catalyse the process of assembly formation. Given this proposal, lamina V cells, in their interaction with ‘library’ cells, would code information in terms of precisely timed individual impulses, but would employ a code based on slower frequency changes in their descending influences upon neural centres in the brainstem and spinal cord. Predictions for single unit and electrographic experiments are discussed. Received: 30 November 1995/Accepted in revised form: 3 June 1996     

Hypocretin (orexin) induces calcium transients in single spines postsynaptic to identified thalamocortical boutons in prefrontal slice

In vivo, thalamocortical axons are susceptible to the generation of terminal spikes which antidromically promote bursting in the thalamus. Although neurotransmitters could elicit such ectopic action potentials at thalamocortical boutons, this hypothesis has never been confirmed. Prefrontal cortex is the cortical area most implicated in arousal and is innervated by thalamic neurons that are unusual since they burst rhythmically during waking. We show that a neurotransmitter critical for alertness, hypocretin (orexin), directly excites prefrontal thalamocortical synapses in acute slice. This TTX-sensitive activation of thalamic axons was demonstrated electrophysiologically and by two-photon sampling of calcium transients at single spines in apposition to thalamic boutons anterogradely labeled in vivo. Spines receiving these long-range projections constituted a unique population in terms of the presynaptic excitatory action of hypocretin. By this mechanism, the hypocretin projection to prefrontal cortex may play a larger role in prefrontal or "executive" aspects of alertness and attention than previously anticipated.     

A thalamo-cortical neural mass model for the simulation of brain rhythms during sleep

Cortico-thalamic interactions are known to play a pivotal role in many brain phenomena, including sleep, attention, memory consolidation and rhythm generation. Hence, simple mathematical models that can simulate the dialogue between the cortex and the thalamus, at a mesoscopic level, have a great cognitive value. In the present work we describe a neural mass model of a cortico-thalamic module, based on neurophysiological mechanisms. The model includes two thalamic populations (a thalamo-cortical relay cell population, TCR, and its related thalamic reticular nucleus, TRN), and a cortical column consisting of four connected populations (pyramidal neurons, excitatory interneurons, inhibitory interneurons with slow and fast kinetics). Moreover, thalamic neurons exhibit two firing modes: bursting and tonic. Finally, cortical synapses among pyramidal neurons incorporate a disfacilitation mechanism following prolonged activity. Simulations show that the model is able to mimic the different patterns of rhythmic activity in cortical and thalamic neurons (beta and alpha waves, spindles, delta waves, K-complexes, slow sleep waves) and their progressive changes from wakefulness to deep sleep, by just acting on modulatory inputs. Moreover, simulations performed by providing short sensory inputs to the TCR show that brain rhythms during sleep preserve the cortex from external perturbations, still allowing a high cortical activity necessary to drive synaptic plasticity and memory consolidation. In perspective, the present model may be used within larger cortico-thalamic networks, to gain a deeper understanding of mechanisms beneath synaptic changes during sleep, to investigate the specific role of brain rhythms, and to explore cortical synchronization achieved via thalamic influences.     

The functional logic of cortico-pulvinar connections

The pulvinar is an 'associative' thalamic nucleus, meaning that most of its input and output relationships are formed with the cerebral cortex. The function of this circuitry is little understood and its anatomy, though much investigated, is notably recondite. This is because pulvinar connection patterns disrespect the architectural subunits (anterior, medial, lateral and inferior pulvinar nuclei) that have been the traditional reference system. This article presents a simplified, global model of the organization of cortico-pulvinar connections so as to pursue their structure-function relationships. Connections between the cortex and pulvinar are topographically organized, and as a result the pulvinar contains a 'map' of the cortical sheet. However, the topography is very blurred. Hence the pulvinar connection zones of nearby cortical areas overlap, allowing indirect transcortical communication via the pulvinar. A general observation is that indirect cortico-pulvino-cortical circuits tend to mimic direct cortico-cortical pathways: this is termed 'the replication principle'. It is equally apt for certain pairs (or groups) of nearby cortical areas that happen not to connect with each other. The 'replication' of this non-connection is achieved by discontinuities and dislocations of the cortical topography within the pulvinar, such that the associated pair of connection zones do not overlap. Certain of these deformations can be used to divide the global cortical topography into specific sub-domains, which form the natural units of a connectional subdivision of the pulvinar. A substantial part of the pulvinar also expresses visual topography, reflecting visual maps in occipital cortex. There are just two well-ordered visual maps in the pulvinar, that both receive projections from area V1, and several other occipital areas; the resulting duplication of cortical topography means that each visual map also acts as a separate connection domain. In summary, the model identifies four topographically ordered connection domains, and reconciles the coexistence of visual and cortical maps in two of them. The replication principle operates at and below the level of domain structure. It is argued that cortico-pulvinar circuitry replicates the pattern of cortical circuitry but not its function, playing a more regulatory role instead. Thalamic neurons differ from cortical neurons in their inherent rhythmicity, and the pattern of cortico-thalamic connections must govern the formation of specific resonant circuits. The broad implication is that the pulvinar acts to coordinate cortical information processing by facilitating and sustaining the formation of synchronized trans-areal assemblies; a more pointed suggestion is that, owing to the considerable blurring of cortical topography in the pulvinar, rival cortical assemblies may be in competition to recruit thalamic elements in order to outlast each other in activity.     

Spatial principle of organization of specific afferent projections and generation of evoked potentials in the somatosensory cortex

Here we investigate the functional organization of structures involved in sensory analysis in a restricted region of a cortical projection area. We have shown that stimulation of somatosensory areas I and II (SI and SII) may block an afferent volley at the level of the thalamic relay nucleus, and that SII may be selectively blocked by stimulation of SI. Also definite somatosensory connections have been demonstrated between SII, SI, and the motor cortex. We suggest that common mechanisms underlie the generation of focal reactions in projection areas of the cortex induced by stimulation of various structures. The properties of two groups of neurones from area SII are described: those having a short latency and receiving direct projections from the thalamic relay nucleus, and those of long latent period with a well-marked convergence, and reacting to stimulation of various afferent pathways. It is suggested that each path to a local point of a cortical projection areas terminates with its relay element. The signal is then directed to a common intracortical system of neurones where signals from various sources occurs (afferent, interhemispherical, subcortico-cortical, and intracortical) converge and interact. All groups of neurones are involved in the formation of the common components of evoked potentials.Presented to the All-Union Symposium: "Electrical responses of the cerebral cortex to afferent stimuli," Kiev, October, 1969.Institute of Normal and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 2, No. 2, pp. 155–165, March–April, 1970.     

A formalisation of the neural assembly concept 1. Constraints on neural assembly size

Hebb proposed the concept of a neural assembly distributed across cortical tissue as a model for representation of information in the cerebral cortex. Later developments of the concept highlight the need for overlapping membership between independent assemblies, and the spread of activity throughout the assembly once it is activated above a critical level (ignition). Formalisation of the neural assembly concept, especially in relation to quantitative data from the real cortex, is at a very early stage. We consider two constraints on neural assembly size: (1) if a neural assembly is too small the fraction of its neurons that need to be active to ignite the whole assembly becomes unrealistically large; (2) if assemblies in a block of cortical tissue become too large then the block becomes ‘unsafe’, that is, unwanted spread from an active assembly to overlapping ones becomes inevitable. We consider variations in three parameters: neuronal firing threshold; connection density; and the total number of assemblies stored in the block of cortical tissue. Given biologically plausible values for these parameters we estimate maximum assembly size compatible with ignitability of individual assemblies, low probability of unwanted spread to overlapping assemblies, and safe operation of the block as a whole. Received: 7 March 1997 / Accepted in revised form: 1 July 1997     

Dendritic spine heterogeneity determines afferent-specific Hebbian plasticity in the amygdala

Functional compartmentalization of dendrites is thought to underlie afferent-specific integration of neural activity in laminar brain structures. Here we show that in the lateral nucleus of the amygdala (LA), an area lacking apparent laminar organization, thalamic and cortical afferents converge on the same dendrites, contacting neighboring but morphologically and functionally distinct spine types. Large spines contacted by thalamic afferents exhibited larger Ca(2+) transients during action potential backpropagation than did small spines contacted by cortical afferents. Accordingly, induction of Hebbian plasticity, dependent on postsynaptic spikes, was restricted to thalamic afferents. This synapse-specific effect involved activation of R-type voltage-dependent Ca(2+) channels preferentially located at thalamic inputs. These results indicate that afferent-specific mechanisms of postsynaptic, associative Hebbian plasticity in LA projection neurons depend on local, spine-specific morphological and molecular properties, rather than global differences between dendritic compartments.     

Characterization of factors regulating lamina‐specific growth of thalamocortical axons

During development, most thalamocortical axons extend through the deep layers to terminate in layer 4 of neocortex. To elucidate the molecular mechanisms that underlie the formation of layer‐specific thalamocortical projections, axon outgrowth from embryonic rat thalamus onto postnatal neocortical slices which had been fixed chemically was used as an experimental model system. When the thalamic explant was juxtaposed to the lateral edge of fixed cortical slice, thalamic axons extended farther in the deep layers than the upper layers. Correspondingly, thalamic axons entering from the ventricular side extended farther than those from the pial side. In contrast, axons from cortical explants cultured next to fixed cortical slices tended to grow nearly as well in the upper as in the deep layers. Biochemical aspects of lamina‐specific thalamic axon growth were studied by applying several enzymatic treatments to the cortical slices prior to culturing. Phosphatidylinositol phospholipase C treatment increased elongation of thalamic axons in the upper layers without influencing growth in the deep layers. Neither chondroitinase, heparitinase, nor neuraminidase treatment influenced the overall projection pattern, although neuraminidase slightly decreased axonal elongation in the deep layers. These findings suggest that glycosylphosphatidylinositol‐linked molecules in the cortex may contribute to the laminar specificity of thalamocortical projections by suppressing thalamic axon growth in the upper cortical layers. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 56–68, 2000     

Characterization of factors regulating lamina-specific growth of thalamocortical axons

During development, most thalamocortical axons extend through the deep layers to terminate in layer 4 of neocortex. To elucidate the molecular mechanisms that underlie the formation of layer-specific thalamocortical projections, axon outgrowth from embryonic rat thalamus onto postnatal neocortical slices which had been fixed chemically was used as an experimental model system. When the thalamic explant was juxtaposed to the lateral edge of fixed cortical slice, thalamic axons extended farther in the deep layers than the upper layers. Correspondingly, thalamic axons entering from the ventricular side extended farther than those from the pial side. In contrast, axons from cortical explants cultured next to fixed cortical slices tended to grow nearly as well in the upper as in the deep layers. Biochemical aspects of lamina-specific thalamic axon growth were studied by applying several enzymatic treatments to the cortical slices prior to culturing. Phosphatidylinositol phospholipase C treatment increased elongation of thalamic axons in the upper layers without influencing growth in the deep layers. Neither chondroitinase, heparitinase, nor neuraminidase treatment influenced the overall projection pattern, although neuraminidase slightly decreased axonal elongation in the deep layers. These findings suggest that glycosylphosphatidylinositol-linked molecules in the cortex may contribute to the laminar specificity of thalamocortical projections by suppressing thalamic axon growth in the upper cortical layers.     

Multiple roles of ephrins during the formation of thalamocortical projections: maps and more

The functional architecture of the cerebral cortex is based on intrinsic connections that precisely link neurons from distinct cortical laminae as well as layer-specific afferent and efferent projections. Experimental strategies using in vitro assays originally developed by Friedrich Bonhoeffer have suggested that positional cues confined to individual layers regulate the assembly of local cortical circuits and the formation of thalamocortical projections. One of these wiring molecules is ephrinA5, a ligand for Eph receptor tyrosine kinases. EphrinA5 and Eph receptors exhibit highly dynamic expression patterns in distinct regions of the cortex and thalamus during early and late stages of thalamocortical and cortical circuit formation. In vitro assays suggest that ephrinA5 is a multifunctional wiring molecule for different populations of cortical and thalamic axons. Additionally, the expression patterns of ephrinA5 during cortical development are consistent with this molecule regulating, in alternative ways, specific components of thalamic and cortical connectivity. To test this directly, the organization of thalamocortical projections was examined in mice lacking ephrinA5 gene expression. The anatomical studies in ephrinA5 knockout animals revealed a miswiring of limbic thalamic projections and changes in neocortical circuits that were predicted from the expression pattern and the in vitro analysis of ephrinA5 function.     

The early topography of thalamocortical projections is shifted in Ebf1 and Dlx1/2 mutant mice

The prevailing model to explain the formation of topographic projections in the nervous system stipulates that this process is governed by information located within the projecting and targeted structures. In mammals, different thalamic nuclei establish highly ordered projections with specific neocortical domains and the mechanisms controlling the initial topography of these projections remain to be characterized. To address this issue, we examined Ebf1(-/-) embryos in which a subset of thalamic axons does not reach the neocortex. We show that the projections that do form between thalamic nuclei and neocortical domains have a shifted topography, in the absence of regionalization defects in the thalamus or neocortex. This shift is first detected inside the basal ganglia, a structure on the path of thalamic axons, and which develops abnormally in Ebf1(-/-) embryos. A similar shift in the topography of thalamocortical axons inside the basal ganglia and neocortex was observed in Dlx1/2(-/-) embryos, which also have an abnormal basal ganglia development. Furthermore, Dlx1 and Dlx2 are not expressed in the dorsal thalamus or in cortical projections neurons. Thus, our study shows that: (1) different thalamic nuclei do not establish projections independently of each other; (2) a shift in thalamocortical topography can occur in the absence of major regionalization defects in the dorsal thalamus and neocortex; and (3) the basal ganglia may contain decision points for thalamic axons' pathfinding and topographic organization. These observations suggest that the topography of thalamocortical projections is not strictly determined by cues located within the neocortex and may be regulated by the relative positioning of thalamic axons inside the basal ganglia.     

Modification of a Colliculo-thalamocortical Mouse Brain Slice,Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice¹, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.     

Parallel driving and modulatory pathways link the prefrontal cortex and thalamus

Pathways linking the thalamus and cortex mediate our daily shifts from states of attention to quiet rest, or sleep, yet little is known about their architecture in high-order neural systems associated with cognition, emotion and action. We provide novel evidence for neurochemical and synaptic specificity of two complementary circuits linking one such system, the prefrontal cortex with the ventral anterior thalamic nucleus in primates. One circuit originated from the neurochemical group of parvalbumin-positive thalamic neurons and projected focally through large terminals to the middle cortical layers, resembling 'drivers' in sensory pathways. Parvalbumin thalamic neurons, in turn, were innervated by small 'modulatory' type cortical terminals, forming asymmetric (presumed excitatory) synapses at thalamic sites enriched with the specialized metabotropic glutamate receptors. A second circuit had a complementary organization: it originated from the neurochemical group of calbindin-positive thalamic neurons and terminated through small 'modulatory' terminals over long distances in the superficial prefrontal layers. Calbindin thalamic neurons, in turn, were innervated by prefrontal axons through small and large terminals that formed asymmetric synapses preferentially at sites with ionotropic glutamate receptors, consistent with a driving pathway. The largely parallel thalamo-cortical pathways terminated among distinct and laminar-specific neurochemical classes of inhibitory neurons that differ markedly in inhibitory control. The balance of activation of these parallel circuits that link a high-order association cortex with the thalamus may allow shifts to different states of consciousness, in processes that are disrupted in psychiatric diseases.     

Functional topography of corticothalamic feedback enhances thalamic spatial response tuning in the somatosensory whisker/barrel system

Corticothalamic (CT) projections are approximately 10 times more numerous than thalamocortical projections, yet their function in sensory processing is poorly understood. In particular, the functional significance of the topographic precision of CT feedback is unknown. We addressed these issues in the rodent somatosensory whisker/barrel system by deflecting individual whiskers and pharmacologically enhancing activity in layer VI of single whisker-related cortical columns. Enhancement of corticothalamic activity in a cortical column facilitated whisker-evoked responses in topographically aligned thalamic barreloid neurons, while activation of an adjacent column weakly suppressed activity at the same thalamic site. Both effects were more pronounced when stimulating the preferred, or principal, whisker than for adjacent whiskers. Thus, facilitation by homologous CT feedback sharpens thalamic receptive field focus, while suppression by nonhomologous feedback diminishes it. Our findings demonstrate that somatosensory cortex can selectively regulate thalamic spatial response tuning by engaging topographically specific excitatory and inhibitory mechanisms in the thalamus.     

Depolarization recruits DCC to the plasma membrane of embryonic cortical neurons and enhances axon extension in response to netrin-1

The netrin-1 receptor Deleted in Colorectal Cancer (DCC) is required for the formation of major axonal projections by embryonic cortical neurons, including the corpus callosum, hippocampal commissure, and cortico-thalamic tracts. The presentation of DCC by axonal growth cones is tightly regulated, but the mechanisms regulating DCC trafficking within neurons are not well understood. Here, we investigated the mechanisms regulating DCC recruitment to the plasma membrane of embryonic cortical neurons. In embryonic spinal commissural neurons, protein kinase A (PKA) activation recruits DCC to the plasma membrane and enhances axon chemoattraction to netrin-1. We demonstrate that PKA activation similarly recruits DCC and increases embryonic cortical neuron axon extension, which, like spinal commissural neurons, respond to netrin-1 as a chemoattractant. We then determined if depolarization might recruit DCC to the plasma membrane. Neither netrin-1 induced axon extension, nor levels of plasma membrane DCC, were altered by depolarizing embryonic spinal commissural neurons with elevated levels of KCl. In contrast, depolarizing embryonic cortical neurons increased the amount of plasma membrane DCC, including at the growth cone, and increased axon outgrowth evoked by netrin-1. Inhibition of PKA, phosphatidylinositol-3-kinase, protein kinase C, or exocytosis blocked the depolarization-induced recruitment of DCC and suppressed axon outgrowth. Inhibiting protein synthesis did not affect DCC recruitment, nor were the distributions of trkB or neural cell adhesion molecule (NCAM) influenced by depolarization, consistent with selective mobilization of DCC. These findings identify a role for membrane depolarization modulating the response of axons to netrin-1 by regulating DCC recruitment to the plasma membrane.     

Organization of the thalamic and cortical projections of the cat neostriatum]

The investigation has demonstrated that in the cat the nucleus caudatus and the putamen are projected on the cortex and thalamic nuclei of the ipsilateral hemisphere according to a certain topical principle characterized by both similarity in localization of projections of these two structures of the neostriatum and their difference. On the one hand, to the same fields of the cortex and the thalamic nuclei fibres from both structures of the neostriatum go, and on the other hand--a number of cortical zones and thalamic nuclei get projections either from the nucleus caudatus or from the putamen only. Owing to a certain organization of the connections studied, it is possible to consider them as the base of functional heterogeneity of the basal ganglia. Over-lapping of the cortical and thalamic projections of the nucleus caudatus and the putamen might explain common striatal effects on behavioral reactions.     

Acetylcholine influences growth cone motility and morphology of developing thalamic axons

The neurotransmitter acetylcholine (ACh) is expressed in the developing telencephalon at the time when thalamic axons project to the cortex, long before synapses are being formed. Since previous studies demonstrated an influence of ACh on neurite extension we used different in vitro assays to examine possible effects of ACh on the growth of thalamic axons. In explant cultures, application of ACh reduced the length of thalamic axons in a dose dependent manner, an effect that could also be evoked by selective muscarinic and nicotinic agonists. Time-lapse imaging of thalamic axons exposed to microscopic gradients of ACh revealed that growth cones no longer advanced, but maintained high filopodial activity. This growth cone pausing was not accompanied by axon retraction or growth cone collapse. It could at least partially be blocked by muscarinic and nicotinic antagonists, indicating that both types of ACh receptors contribute to mediate these effects on thalamic axons. Finally, we also found that ACh changed the morphology of growth cones; they became larger and extended more filopodia. Since such changes in the structure and motility of growth cones are observed at decision regions along the path of many fiber populations including thalamic axons, we suggest that ACh plays a role during the elaboration of thalamocortical projections.Key words: cortical development, thalamocortical projections, neurotransmitter, acetylcholine, growth cone, axonal guidance, wiring molecules     

Visual thalamic afferents to area 29 of the rat limbic cortex

Afferent connections of the retrosplenial area of the rat limbic cortex were investigated by the retrograde horseradish peroxidase axon transport method. After injection of horseradish peroxidase (HRP) into area 29 of the cortex, HRP-labeled cells were found in the dorsal part of the lateral geniculate body and the posterolateral, pretectal, and anterior dorsal thalamic nuclei. Connections were found between cortical area 29 and visual projection areas (areas 17 and 18a) and with area 29 on the contralateral side of the brain. The results are evidence that all the principal visual structures of the thalamus and the visual cortical projection area form direct projections to the retrosplenial cortex.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 14, No. 2, pp. 135–139, March–April, 1982.     

Specificity of projection among cells of the zona incerta

We have examined whether individual cells of the zona incerta of the thalamus have widespread projections across the brain. Double injections of different coloured fluorescent latex beads (red or green) were made, in various combinations, into regions of neocortex, dorsal thalamus or brainstem of Sprague-Dawley rats. These regions were chosen since they have been shown previously to receive projections from the zona incerta. We also made injections of different coloured beads into different regions of these same brain centres (ie, distinct cortical areas or individual dorsal thalamic and brainstem nuclei). In general, our results show that cells of the zona incerta have projections limited to one of these brain centres only. We saw very few double-labelled incertal cells after double injections of different coloured latex beads into either the neocortex/dorsal thalamus, neocortex/brainstem or dorsal thalamus/brainstem. Further, we show that within each of these brain centres, the projection patterns of individual incertal cells is rather restricted, since double injections of different coloured beads into separate regions of the same centre resulted in few double-labelled incertal cells. Taken together, these results suggest a very clear specificity of projection among cells of the zona incerta. Thus, although the cells of the zona incerta receive a plethora of inputs from many sources, it appears that its cells have a very clear and focussed output to distinct regions of the brain.     

Comparison of the Connectional Properties of the Two Forelimb Areas of the Rat Sensorimotor Cortex: Support for the Presence of a Premotor or Supplementary Motor Cortical Area

The existence of multiple motor cortical areas that differ in some of their properties is well known in primates, but is less clear in the rat. The present study addressed this question from the point of view of connectional properties by comparing the afferent and efferent projections of the caudal forelimb area (CFA), considered to be the equivalent of the forelimb area of the primary motor cortex (MI), and a second forelimb motor representation, the rostral forelimb area (RFA). As a result of various tracing experiments (including double labeling), it was observed that CFA and RFA had reciprocal corticocortical connections characterized by preferential, asymmetrical, laminar distribution, indicating that RFA may occupy a different hierarchical level than CFA, according to criteria previously discussed in the visual cortex of primates. Furthermore, it was found that RFA, but not CFA, exhibited dense reciprocal connections with the insular cortex. With respect to their efferent projection to the basal ganglia, it was observed that CFA projected very densely to the lateral portion of the ipsilateral caudate putamen, whereas the contralateral projection was sparse and more restricted. The ipsilateral projection originating from RFA was slightly less dense than that from CFA, but it covered a larger portion of the caudate putamen (in the medial direction); the contralateral projection from RFA to the caudate putamen was of the same density and extent as the ipsilateral projection. The reciprocal thalamocortical and corticothalamic connections of RFA and CFA differed from each other in the sense that CFA was mainly interconnected with the ventrolateral thalamic nucleus, while RFA was mainly connected with the ventromedial thalamic nucleus. Altogether, these connectional differences, compared with the pattern of organization of the motor cortical areas in primates, suggest that RFA in the rat may well be an equivalent of the premotor or supplementary motor area. In contrast to the corticocortical, corticostriatal, and thalamocortical connections, RFA and CFA showed similar efferent projections to the subthalamic nucleus, substantia nigra, red nucleus, tectum, pontine nuclei, inferior olive, and spinal cord.