Collateral Axonal Branching Research Articles

Electrical stimulation of paralyzed vibrissal muscles reduces endplate reinnervation and does not promote motor recovery after facial nerve repair in rats

The outcome of peripheral nerve injuries requiring surgical repair is poor. Recent work has suggested that electrical stimulation (ES) of denervated muscles could be beneficial. Here we tested whether ES has a positive influence on functional recovery after injury and surgical repair of the facial nerve. Outcomes at 2 months were compared to animals receiving sham stimulation (SS). Starting on the first day after end-to-end suture (facial-facial anastomosis), electrical stimulation (square 0.1 ms pulses at 5 Hz at an ex tempore established threshold amplitude of between 3.0 and 5.0V) was delivered to the vibrissal muscles for 5 min a day, 3 times a week. Restoration of vibrissal motor performance following ES or SS was evaluated using the video-based motion analysis and correlated with the degree of collateral axonal branching at the lesion site, the number of motor endplates in the target musculature and the quality of their reinnervation, i.e. the degree of mono- versus poly-innervation. Neither protocol reduced collateral branching. ES did not improve functional outcome, but rather reduced the number of innervated motor endplates to approximately one-fifth of normal values and failed to reduce the proportion of poly-innervated motor endplates. We conclude that ES is not beneficial for recovery of whisker function after facial nerve repair in rats.

Schwann Cells Overexpressing FGF‐2 Alone or Combined with Manual Stimulation Do Not Promote Functional Recovery after Facial Nerve Injury

Purpose. To determine whether transplantation of Schwann cells (SCs) overexpressing different isoforms of fibroblast growth factor 2 (FGF-2) combined with manual stimulation (MS) of vibrissal muscles improves recovery after facial nerve transection in adult rat. Procedures. Transected facial nerves were entubulated with collagen alone or collagen plus naïve SCs or transfected SCs. Half of the rats received daily MS. Collateral branching was quantified from motoneuron counts after retrograde labeling from 3 facial nerve branches. Quality assessment of endplate reinnervation was combined with video-based vibrissal function analysis. Results. There was no difference in the extent of collateral axonal branching. The proportion of polyinnervated motor endplates for either naïve SCs or FGF-2 over-expressing SCs was identical. Postoperative MS also failed to improve recovery. Conclusions. Neither FGF-2 isoform changed the extent of collateral branching or polyinnervation of motor endplates; furthermore, this motoneuron response could not be overridden by MS.

Manual Stimulation of the Suprahyoid-Sublingual Region Diminishes Polynnervation of the Motor Endplates and Improves Recovery of Function After Hypoglossal Nerve Injury in Rats

Background. Using the rat facial nerve axotomy model, the authors recently showed that manual stimulation of denervated whiskerpad muscles reduced the posttransectional polyinnervation at the neuromuscular junctions and promoted full recovery of vibrissal whisking. Objective. Prompted by implications for rehabilitation therapy, the authors examined whether manual stimulation of denervated supra- and infrahyoid muscles would also improve recovery after unilateral lesion on the hypoglossal nerve. Methods. Adult rats underwent transection of the right hypoglossal nerve. Half of the animals received no postoperative treatment, and the other half were subjected to daily manual stimulation of the suprahyoid/sublingual region for 2 months. Recovery was assessed by measuring the angle of tongue-tip deviation from the midline, degree of collateral axonal branching at the lesion site (counts after retrograde labeling with 2 fluorescent dyes), synaptic input to the hypoglossal motoneurons using synaptophysin immunocytochemistry, tongue-muscles motor representation in the cerebral cortex after c-Fos immunocytochemistry, and portion of polyinnervated neuromuscular junctions. Results. In animals receiving manual stimulation, the tongue-tip deviation was 37.0 ± 49.37°, whereas values in control nonstimulated rats were significantly higher (50.1 ± 9.01°; P < .05; mean ± SD). Improved recovery was not associated with reduced collateral axonal branching; there were also no differences in tongue-muscles representation in the motor cortex. However, manual stimulation restored the total synaptic input to levels in intact animals and reduced the proportion of polyinnervated neuromuscular junctions compared with nonstimulated animals. Conclusion. The data show that manual stimulation of denervated muscles improves functional outcome following peripheral nerve injury. This suggests immediate potential for enhancing clinical rehabilitation strategies.

Bone marrow-derived mesenchymal stem cell transplantation does not improve quality of muscle reinnervation or recovery of motor function after facial nerve transection in rats

Recently, we devised and validated a novel strategy in rats to improve the outcome of facial nerve reconstruction by daily manual stimulation of the target muscles. The treatment resulted in full recovery of facial movements (whisking), which was achieved by reducing the proportion of pathologically polyinnervated motor endplates. Here, we posed whether manual stimulation could also be beneficial after a surgical procedure potentially useful for treatment of large peripheral nerve defects, i.e., entubulation of the transected facial nerve in a conduit filled with suspension of isogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) in collagen. Compared to control treatment with collagen only, entubulation with BM-MSCs failed to decrease the extent of collateral axonal branching at the lesion site and did not improve functional recovery. Post-operative manual stimulation of vibrissal muscles also failed to promote a better recovery following entubulation with BM-MSCs. We suggest that BM-MSCs promote excessive trophic support for regenerating axons which, in turn, results in excessive collateral branching at the lesion site and extensive polyinnervation of the motor endplates. Furthermore, such deleterious effects cannot be overridden by manual stimulation. We conclude that entubulation with BM-MSCs is not beneficial for facial nerve repair.

Collateral projections from the subfornical organ to the median preoptic nucleus and paraventricular hypothalamic nucleus in the rat

It is morphologically demonstrated that the subfornical organ (SFO) projects to the paraventricular hypothalamic nucleus (PVN) and also projects to the nucleus preopticus medianus (POMe), a relay nucleus of indirect projections from the SFO to PVN. However, it remains unknown, whether or not SFO neurons project collaterally to the POMe and PVN. To confirm this, a double retrograde labeling method was performed on rats using two fluorescent tracers. One tracer (red-colored FluoSpheres: FSR) was injected into the POMe and the other (Fast Blue: FB) was injected into the unilateral PVN at the same time. As a result, many retrogradely labeled neurons were found in the entire SFO. Of these, some neurons showed both FSR and FB fluorescence. Double-labeled neurons were found in about 8.7% of FSR-labeled neurons and 15.5% of FB-labeled neurons. The existence of double-labeled neurons indicates that single neurons in the SFO project simultaneously to the POMe and PVN via collateral axon branches. The data suggest that there are complicated neuronal pathways originating from the SFO in regulating cardiovascular and body fluid homeostasis.

IMPACT OF DIFFERENT TYPES OF FACIAL NERVE RECONSTRUCTION ON THE RECOVERY OF MOTOR FUNCTION

Poor functional recovery after facial nerve reconstruction is characterized by mass movements and synkinesis. Major reasons are axonal sprouting from the regenerating axons leading to misdirected reinnervation and hyperinnervation as well as polyinnervation of the mimic muscle end plates. We analyzed whether or not the type of nerve reconstruction influenced these pathological phenomena. The experiments were performed on 48 adult rats divided into four groups. One group served as an intact control and the experimental groups were subjected to facial-facial nerve repair (FFN), facial nerve interpositional grafting, and hypoglossal-facial nerve repair (HFN), with 12 subjects in each group. Two months later, functional recovery was measured by biometrical motion analysis of whisking. Retrograde fluorescence labeling of the brainstem motoneurons was used to quantify the degree of collateral axonal branching at the lesion site. Fluorescence histochemistry of sections through the levator labii superioris muscle was performed to quantify the degree of polyinnervation after surgery. The type of nerve reconstruction significantly influenced the regeneration. The whisking amplitude did not recover completely regardless of the type of reconstruction. The angular velocity and angular acceleration of the vibrissal hairs showed a full recovery after facial nerve interpositional grafting and HFN, whereas these parameters remained decreased after FFN. Significantly less collateral branching and polyinnervation of the end plates were determined after grafting and HFN than after FFN. No type of immediate facial nerve reconstruction results in a full recovery in the rat. However, the morphological and functional recovery was significantly better after grafting and HFN than after FFN.

Local stabilization of microtubule assembly improves recovery of facial nerve function after repair

Within a recent study on the recovery of vibrissae motor performance after facial nerve repair in blind (strain SD/RCS) and sighted (strain SD) rats, we found that, despite persisting myotopic disorganization in the facial nucleus, the blind animals fully restored vibrissal whisking. Searching for the morphological substrates of this improved recovery, we compared the amount of cytoskeletal proteins in the leading edge of elongating axons between both strains. Since our results showed an enhanced expression of neuronal class III β-tubulin in the blind rats, we wondered whether this was due to an increased synthesis or to a delayed turnover of microtubules. In the present report, we approached this question applying established pharmacological agents to the transected buccal branch of the facial nerve in sighted Wistar rats perturbing either microtubule assembly towards stabilization (enhanced polymerization with 10 μg/ml taxol) or towards increased synthesis (challenged by destabilization with 100 μg/ml nocodazole and 20 μg/ml vinblastine). Evaluation of the effect(s) 2 months later included estimation of (i) vibrissae motor performance by video-based motion analysis, (ii) the degree of collateral axonal branching by double retrograde neuronal labeling with crystals of Fluoro-Gold and DiI and (iii) the pattern of motor end-plate reinnervation (proportions of mono- and poly-reinnervated) in the largest extrinsic vibrissal muscle, the m. levator labii superioris. We found that only stabilization of microtubules with 10 μg/ml taxol reduced intramuscular axonal sprouting and polyinnervation of the motor end-plates, which was accompanied by improved restoration of function.

The axotomy‐induced neuropeptides galanin and pituitary adenylate cyclase‐activating peptide promote axonal sprouting of primary afferent and cranial motor neurones

The neuropeptides galanin and pituitary adenylate cyclase-activating peptide (PACAP) are markedly up-regulated in response to peripheral nerve lesion. Both peptides are involved in neuronal differentiation and neurite outgrowth during development. In this study, we investigated the effects of galanin and PACAP on axonal elongation and sprouting by adult rat sensory neurones in vitro and facial motor neurones in vivo. Dissociated rat dorsal root ganglion neurones were plated on laminin substrate and analysed morphometrically. Both the mean axonal length and the number of branch points significantly increased in the presence of galanin or PACAP (2-5 microm). Effects on axonal collateralization were investigated in the rat facial nerve lesion model by direct application of the peptides to collagen-filled conduits entubulating the transected facial nerve stumps. Triple retrograde labelling of brainstem neurones confirmed that the peptides potently induce axonal sprouting of cranial motor neurones. The number of neurones regenerating into identified rami of the facial nerve increased up to fivefold. Biometrical analysis of whisking behaviour revealed that galanin and PACAP impaired the functional outcome when compared with vehicle-treated animals 8 weeks after surgery. In conclusion, although galanin and PACAP have been established as neurotrophic molecules with respect to axonal development and regeneration, their potential as treatments for peripheral nerve lesions appears limited because of the extensive stimulation of collateral axon branching. These branches are misrouted towards incorrect muscles and cause impairment in their coordinated activity.

Genetic Mechanisms Specifying Cortical Connectivity

Great neuroanatomists of the twentieth century recognized that the cerebral cortex of mammals is the single most complex structure of the central nervous system both in terms of neuronal diversity and connectivity. Understanding the cellular and molecular mechanisms specifying the afferent and efferent connectivity in the neocortex may seem like a daunting task. However, recent technical advances have greatly improved our ability to (1) profile gene expression of neuronal populations isolated based on their connectional properties, (2) manipulate gene expression in specific neuronal populations, and (3) visualize their axonal projections in vivo. These new tools are revolutionizing our ability to identify the molecular mechanisms patterning afferent and efferent cortical projections.

Olfactory ensheathing cells promote collateral axonal branching in the injured adult rat spinal cord

In recent years, injection of olfactory ensheathing cells (ECs) into the spinal cord has been used as an experimental strategy to promote regeneration of injured axons. In this study, we have compared the effects of transplanting encapsulated ECs with those injected directly into the spinal cord. The dorsal columns of adult rats were cut at T 8–9 and rats in experimental groups received either EC-filled porous polymer capsules or culture medium (CM)-filled capsules with ECs injected at the injury site. Control rats were in three groups: (1) uninjured, (2) lesion with transplantation of CM-filled capsules and (3) lesion with transplantation of CM-filled capsules and injections of CM. Three weeks after injury, Fluororuby was injected into the hindlimb motor and somatosensory cortex to label corticospinal neurons. Observations indicated that there were a few regenerating fibres, up to 10, in the EC-treated groups. In rats that received encapsulated ECs, regenerating fibres were present in close association with the capsule. Rats that received EC injections demonstrated a significant increase in the number of collateral branches from the intact ventral corticospinal tract (vCST) compared with the corresponding control, CM-injected group ( P = 0.003), while a trend for increased collateral branches was observed in rats that received encapsulated ECs ( P = 0.07).

Reduced rates of axonal and dendritic growth in embryonic hippocampal neurones cultured from a mouse model of Sandhoff disease.

Sandhoff disease is a lysosomal storage disease in which ganglioside GM2 accumulates because of a defective beta-subunit of beta-hexosaminidase. This disease is characterized by neurological manifestations, although the pathogenic mechanisms leading from GM2 accumulation to neuropathology are largely unknown. We now examine the viability, development and rates of neurite growth of embryonic hippocampal neurones cultured from a mouse model of Sandhoff disease, the Hexb-/- mouse. GM2 was detected by metabolic labelling at low levels in wild type (Hexb+/+) neurones, and increased by approximately three-fold in Hexb-/- neurones. Hexb-/- hippocampal neurones were as viable as their wild type counterparts and, moreover, their developmental programme was unaltered because the formation of axons and of the minor processes which eventually become dendrites was similar in Hexb-/- and Hexb+/+ neurones. In contrast, once formed, a striking difference in the rate of axonal and minor process growth was observed, with changes becoming apparent after 3 days in culture and highly significant after 5 days in culture. Analysis of various parameters of axonal growth suggested that a key reason for the decreased rate of axonal growth was because of a decrease in the formation of collateral axonal branches, the major mechanism by which hippocampal axons elongate in culture. Thus, although the developmental programme with respect to axon and minor process formation and the viability of hippocampal neurones are unaltered, a significant decrease occurs in the rate of axonal and minor process growth in Hexb-/- neurones. These results appear to be in contrast to dorsal root ganglion neurones cultured from 1-month-old Sandhoff mice, in which cell survival is impaired but normal outgrowth of neurones occurs. The possible reasons for these differences are discussed.

Focal application of neutralizing antibodies to soluble neurotrophic factors reduces collateral axonal branching after peripheral nerve lesion.

A major reason for the insufficient recovery of function after motor nerve injury are the numerous axonal branches which often re-innervate muscles with completely different functions. We hypothesized that a neutralization of diffusable neurotrophic factors at the lesion site in rats could reduce the branching of transected axons. Following analysis of local protein expression by immunocytochemistry and by in situ hybridization, we transected the facial nerve trunk of adult rats and inserted both ends into a silicon tube containing (i) collagen gel with neutralizing concentrations of antibodies to NGF, BDNF, bFGF, IGF-I, CNTF and GDNF; (ii) five-fold higher concentrations of the antibodies and (iii) combination of antibodies. Two months later, retrograde labelling was used to estimate the portion of motoneurons the axons of which had branched and projected into three major branches of the facial trunk. After control entubulation in collagen gel containing non-immune mouse IgG 85% of all motoneurons projecting along the zygomatic branch sprouted and sent at least one twin axon to the buccal and/or marginal-mandibular branches of the facial nerve. Neutralizing concentrations of anti-NGF, anti-BDNF and anti-IGF-I significantly reduced sprouting. The most pronounced effect was achieved after application of anti-BDNF, which reduced the portion of branched neurons to 18%. All effects after a single application of antibodies were concentration-dependent and superior to those observed after combined treatment. This first report on improved quality of reinnervation by antibody-therapy implies that, in rats, the post-transectional collateral axonal branching can be reduced without obvious harmful effects on neuronal survival and axonal elongation.

Axon Branching Requires Interactions between Dynamic Microtubules and Actin Filaments

Cortical neurons innervate many of their targets by collateral axon branching, which requires local reorganization of the cytoskeleton. We coinjected cortical neurons with fluorescently labeled tubulin and phalloidin and used fluorescence time-lapse imaging to analyze interactions between microtubules and actin filaments (F-actin) in cortical growth cones and axons undergoing branching. In growth cones and at axon branch points, splaying of looped or bundled microtubules is accompanied by focal accumulation of F-actin. Dynamic microtubules colocalize with F-actin in transition regions of growth cones and at axon branch points. In contrast, F-actin is excluded from the central region of the growth cone and the axon shaft, which contains stable microtubules. Interactions between dynamic microtubules and dynamic actin filaments involve their coordinated polymerization and depolymerization. Application of drugs that attenuate either microtubule or F-actin dynamics also inhibits polymerization of the other cytoskeletal element. Importantly, inhibition of microtubule or F-actin dynamics prevents axon branching but not axon elongation. However, these treatments do cause undirected axon outgrowth. These results suggest that interactions between dynamic microtubules and actin filaments are required for axon branching and directed axon outgrowth.

Fibroblast Growth Factor-2 Promotes Axon Branching of Cortical Neurons by Influencing Morphology and Behavior of the Primary Growth Cone

Interstitial branching is an important mechanism for target innervation in the developing CNS. A previous study of cortical neurons in vitro showed that the terminal growth cone pauses and enlarges in regions from which interstitial axon branches later develop (Szebenyi et al., 1998). In the present study, we investigated how target-derived signals affect the morphology and behaviors of growth cones leading to development of axon branches. We used bath and local application of a target-derived growth factor, FGF-2, on embryonic pyramidal neurons from the sensorimotor cortex and used time-lapse digital imaging to monitor effects of FGF-2 on axon branching. Observations of developing neurons over periods of several days showed that bath-applied FGF-2 significantly increased growth cone size and slowed growth cone advance, leading to a threefold increase in axon branching. FGF-2 also had acute effects on growth cone morphology, promoting rapid growth of filopodia within minutes. Application of FGF-2-coated beads promoted local axon branching in close proximity to the beads. Branching was more likely to occur when the FGF-2 bead was on or near the growth cone, suggesting that distal regions of the axon are more responsive to FGF-2 than other regions of the axon shaft. Together, these results show that interstitial axon branches can be induced locally through the action of a target-derived growth factor that preferentially exerts effects on the growth cone. We suggest that, in target regions, growth factors such as FGF-2 and other branching factors may induce formation of collateral axon branches by enhancing the pausing and enlargement of primary growth cones that determine future branch points.

Bidirectional Neurones in the Cervical Enlargement of the Cat Spinal Cord with Axons Descending to Sacral Segments and Ascending to the Cerebellum and the Lateral Reticular Nucleus

Neurones located in cervical segments (C6/C7) of the spinal cord were investigated electrophysiologically in cats deeply anaesthetized with alpha-chloralose. Extracellular recordings of antidromic action potentials were performed in order to establish whether long descending propriospinal neurones projecting to sacral segments could have collateral axonal branches ascending to supraspinal centres. The effects of stimulation of the lateral reticular nucleus (LRN) and the inferior cerebellar peduncle (restiform body, RB), as well as the thirteenth thoracic (Th13) and sacral (S1/S2) segments of the spinal cord were tested in 93 cells. Two main groups of cells were identified: 54 % of the total sample were classified as purely propriospinal and 46 % as bidirectional neurones. Various patterns of projections, as well as the ipsi-, contra- or bilateral courses of axons in the lateral funiculi of the spinal cord, enabled several types of neurones to be distinguished within the above groups. Comparison between particular types showed no significant difference with respect to location in the grey matter (predominantly Rexed's laminae VII-VIII) and the conduction velocities of descending axons. However, the mean axonal conduction velocities of branches ascending to LRN and/or RB were significantly lower in comparison to those measured for spinal collaterals. The hypothetical function of the neurones examined is discussed. Since the same information can be conveyed simultaneously by these branching neurones to lower spinal segments and supraspinal centres, an integrative role in the system of motor control is suggested.

Fluorescent double-label study of lateral reticular nucleus projections to the spinal cord and periaqueductal gray in the rat.

Following injections of WGA-HRP into either the spinal cord or periaqueductal gray, labeled neurons were observed bilaterally along the periphery of the lateral reticular nucleus (LRN) magnocellular division. The possibility that some of these neurons in the LRN provide collateral axonal branches to both the periaqueductal gray and the spinal cord was investigated in rats using a retrograde double-labeling method employing two different fluorescent tracers, True Blue and Nuclear Yellow. Following sequential injection of the two fluorescent axonal tracers into the spinal cord and periaqueductal gray in the same animal, a modest number of double-labeled neurons were observed bilaterally near the medial and dorsal perimeter of the magnocellular division of the LRN. The labeled neurons were distinctly multipolar in shape and measured approximately 15-18 mu in their greatest transverse diameter. No double-labeled neurons were observed in the parvocellular or subtrigeminal divisions of the LRN. Based upon these observations, it is suggested that collaterals of the LRN-spinal pathway provide feedback information to the periaqueductal gray that might then be used to modulate the participation of the latter cell group in a variety of pain processing and cardiovascular regulatory functions.

Fluorescent double‐label study of lateral reticular nucleus projections to the spinal cord and periaqueductal gray in the rat

Following injections of WGA-HRP into either the spinal cord or periaqueductal gray, labeled neurons were 7observed bilaterally along the periphery of the lateral reticular nucleus (LRN) magnocellular division. The possibility that some of these neurons in the LRN provide collateral axonal branches to both the periaqueductal gray and the spinal cord was investigated in rats using a retrograde double-labeling method employing two different fluorescent tracers, True Blue and Nuclear Yellow. Following sequential injection of the two fluorescent axonal tracers into the spinal cord and periaqueductal gray in the same animal, a modest number of double-labeled neurons were observed bilaterally near the medial and dorsal perimeter of the magnocellular division of the LRN. The labeled neurons were distinctly multipolar in shape and measured approximately 15–18 μ in their greatest transverse diameter. No double-labeled neurons were observed in the parvocellular or subtrigeminal divisions of the LRN. Based upon these observations, it is suggested that collaterals of the LRN-spinal pathway provide feedback information to the periaqueductal gray that might then be used to modulate the participation of the latter cell group in a variety of pain processing and cardiovascular regulatory functions. Anat Rec 256:91–98, 1999. © 1999 Wiley-Liss, Inc.

Localized Sources of Neurotrophins Initiate Axon Collateral Sprouting

The sprouting of axon collateral branches is important in the establishment and refinement of neuronal connections during both development and regeneration. Collateral branches are initiated by the appearance of localized filopodial activity along quiescent axonal shafts. We report here that sensory neuron axonal shafts rapidly sprout filopodia at sites of contact with nerve growth factor-coated polystyrene beads. Some sprouts can extend up to at least 60 micro(m) through multiple bead contacts. Axonal filopodial sprouts often contained microtubules and exhibited a debundling of axonal microtubules at the site of bead-axon contact. Cytochalasin treatment abolished the filopodial sprouting, but not the accumulation of actin filaments at sites of bead-axon contact. The axonal sprouting response is mediated by the trkA receptor and likely acts through a phosphoinositide-3 kinase-dependent pathway, in a manner independent of intracellular Ca2+ fluctuations. These findings implicate neurotrophins as local cues that directly stimulate the formation of collateral axon branches.

Autosomal dominant distal spinal muscular atrophy in four generations

Distal spinal muscular atrophy is a rare lower motor neuron disorder that may be difficult to distinguish clinically from type II Charcot-Marie-Tooth disease. We report on clinical and pathologic findings in 13 members of a four-generation extended family with autosomal dominant distal spinal muscular atrophy. The patients developed a slowly progressive lower motor neuron disorder involving mainly the distal lower extremities; onset was from the second to fourth decades. Electromyography and muscle biopsy findings were indicative of motor denervation. Combined silver/cholinesterase/immunocytochemical staining of intramuscular nerve revealed abundant collateral axonal branching in mild disease but marked loss of terminal motor endplate innervation in the more severe state, suggesting decreased growth of motor axon collaterals with disease progression. Multipoint DNA linkage analysis showed that this family's disorder is not linked to the chromosome 5q11.2-13.3 spinal muscular atrophy locus.

Patterns of connectivity in the cat sensory‐motor cortex: A light and electron microscope analysis of the projection arising from area 3a

Through its complex network of interconnections, each of the functionally specialized subdivisions in the cat primary somatosensory cortex may provide the motor cortex with different input and thus play a unique role in motor behavior. Areas 2 and 3a receive separate peripheral information. Cells in both regions project directly to the cat motor cortex but are thought to target different populations of neurons. In this study the morphology and distribution patterns of the area 3a projection to the motor cortex were compared to previous findings on the projection from area 2. Also, details of the projection from area 3a to area 2 were studied. Injections of Phaseolus vulgaris leucoagglutinin were made into area 3a and fixed brain tissue was processed for immunohistochemical staining of this anterograde tracer. Tissue was examined with the light microscope to determine the patterns of the 3a projections to area 2 and motor cortex, area 4. Axons arising from cells in area 3a terminated in multiple column-like clusters in both motor cortex and area 2. The small number of cells labeled at the injection sites suggested that multiple foci of the 3a fibers are formed by collateral axonal branches of the same neurons. The topography of the projection from area 3a to the motor cortex was more clearly defined than that from area 3a to area 2. Electron microscope analysis determined the laminar distribution and types of synapses formed between area 3a efferents and their target neurons. A high proportion of synapses was found in layer III in both target regions. However, unlike in area 4, labeled terminals were sparse in laminae I and II of area 2. Axospinous synapses were slightly more numerous than axodendritic synapses, but both were distributed similarly throughout the thickness of the cortex. In area 2 the axospinous synapses accounted for 63% of synapses and in area 4 for 57% of synapses. No axosomatic synapses were detected.