Histological and ultrastructural characterization of interfascicular neurons in the rat anterior commissure

Brain Research 931 (2002) 81–91 www.elsevier.com / locate / bres

Research report

Histological and ultrastructural characterization of interfascicular neurons in the rat anterior commissure ´ Lara, Gema Martınez-Cabrera, ´ Jorge Larriva-Sahd*, Miguel Condes Alfredo Varela-Echavarria ´ , Campus UNAM-UAQ , Apartado Postal 1 -1141, Zona Centro Queretaro ´ , CP 76001, Qro., Mexico Centro de Neurobiologıa Accepted 17 December 2001 This study is dedicated in memoriam to Professor Daniel C. Pease, who died on 7th May 2001.

Abstract The histological, connectional, and ultrastructural characteristics of a peculiar neuron type in the rat anterior commissure (AC) are described. Since these cells are located among the axonal fascicles of the rostral and caudal parts of the AC, they are termed interfascicular neurons (IFN). In rapid-Golgi sections IFNs appeared in two forms: internuncial (i.e., short axon) and projection neurons (i.e., long axon). The axon of the internuncial neurons terminates upon neighboring IFNs. The projection neurons give rise to an axon which is either incorporated into commissural fibers or ramifies into 12–26 collaterals running laterally in opposite directions along commissural axons. Immunohistochemistry to microtubule-associated protein 2 combined with confocal microscopy showed that IFNs display short varicose dendrites which remain confined to the domain of the AC. The neuronal nature of IFNs was confirmed with the electron microscope on the basis of distinctive organelles and the presence of synaptic inputs. Small areas of neuropil surround some IFNs. These areas are composed of proximal dendrites, terminal axons, axo-shaft and axo-spinous synapses. Because IFNs with their afferents and efferents constitute sufficient elements to integrate neural inputs, it is proposed that they may be involved in processing nerve impulses proceeding from the bilateral cerebral structures connected by the AC.  2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Other systems of the CNS, comparative neuroanatomy Keywords: Cytology; Immunohistochemestry; Golgi method; Ultrastructure

1. Introduction Exchange of information between the cerebral hemispheres in mammals relies upon the commissural fiber systems. Projecting neurons from specific forebrain areas are reciprocally connected by three major commissural systems: corpus callosum, hippocampal commissure, and ´ y Cajal anterior commissure (AC) [4,25]. In fact, Ramon early noted that the AC is the main route that interconnects the basal temporal cortex and main olfactory areas of either side of the brain [25]. Since this pioneer description it has become evident that the rodent’s AC is composed of three main axon bundles arranged into two broad but *Corresponding author. Tel.: 152-5-623-4030; fax: 152-4-234-0344. E-mail address: [email protected] (J. Larriva-Sahd).

distinctive areas: a rostral, olfactory, or bulbar part (rAC) and a caudal, temporal, or sphenoidal part (cAC). The rAC contains axons arising from the anterior olfactory nucleus and other olfactory cortical areas [14]. The cAC receives fibers from both banks of the rhinal sulcus in the temporal neocortex [5,25], the main olfactory cortical areas, and the amygdala [4]. Both cortical and posterior nuclei of the amygdala send fibers to the AC [3]. Although each commissural component has the same basic cytological organization composed of axons, oligodendrocytes, astrocytes, and microglia, differences in axon caliber [25] and size of the oligodendrocytes [13] have justified the parceling into rAC and cAC [7,9]. Most brain areas known to project through the AC have been implicated in reproductive, behavioral, and vegetative functions. Thus, attention has been paid to possible structural differences in

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the AC with reference to these. For instance, structural differences in the AC have been found in relation to gender [1,2,10,23,32], sexual orientation [2], drug abuse [24,32], prenatal stress [7], gonadal [13] and thyroid hormone manipulations [4], and development [4,10,27]. Three structural aspects have been evaluated in these studies: the AC area determined in coronal and / or sagittal sections, the structure and number of its constituent axons, and axonal transport of tract-tracing substances. The present study derives from the unexpected observation of neurons and synaptic terminals among the axons of the rat ´ y Cajal first noted the presence of AC. While Ramon long-axon neurons in the cerebellar white matter and fornix longus (i.e., interstitial cells) [25] and Sturrock documented the presence of commissural neurons in the mouse AC with the conventional light and electron microscopy [29], no information is available about either the cytological organization or the interactions of neurons in the rat AC. Some results of the present study were published in an abstract [12].

2. Materials and methods

2.1. Light and electron microscopy Twenty Sprague–Dawley male rats killed at 12 weeks of age were used. The animals were kept under constant temperature (24 8C) and photoperiods (14 h light–10 h dark), with free access to food and water. All experimental manipulations and killings were made according to the ethical policies of animal care and handling of our institute. All rats were killed by transcardial perfusion as previously described [11]. Under deep barbiturate anesthesia (20 mg of pentobarbital per 250 g of body weight) animals were perfused through the left ventricle with a total of 250 ml of Karnovsky’s fixative (4% paraformaldehayde–2% glutaraldehyde in 0.15 M sodium cacodylate buffer, pH 7.3). The brain was then removed and transferred to fresh 4 8C fixative and left overnight. The following morning each brain was serially sectioned at a thickness of 200 mm with a vibrotome. Nine brains were sectioned in the sagittal plane and ten in the coronal plane, as close as possible to the orientation of tissue blocks with the planes described elsewhere [8]. The AC was removed from the appropriate sections under a dissecting microscope. The lateral boundary of the AC was determined at the lateral point where axons of the decussating component of the stria terminalis have been incorporated to the AC. This boundary corresponds to a vertical 1.5 mm from the sagittal plane as described by Kruger et al. [8]. The tissue samples were postfixed for 1 h in 1% osmium tetraoxide dissolved in the same buffer as the aldehydes, dehydrated, and flat embedded in epoxy resins. One micrometer thin sections were obtained from the tissue blocks in a Leica ultramicrotome equipped with glass knives. The sections

were stained with toluidine blue and cover-slipped. From the surface of these trimmed blocks, ultra-thin sections ranging from 80 to 90 nm were obtained with a diamond knife and mounted in single slot grids, which had previously been covered with formvar film. The sections were double stained with aqueous solutions of uranium acetate and lead citrate, and observed in a JEOL 1010 electron microscope. In order to estimate the approximate occurrence of neurons in the AC, one brain was taken at random and blocked free-hand with a razor blade in parallel coronal cuts aided by a stainless steel slicing device (Pellco, Tustin, CA, USA). The resulting tissue block was processed as described above and sectioned throughout the entire AC, as follows: A 1-mm thin section from every 20 was mounted, stained with toluidine blue and studied under the light microscope. The incidence and location of each neuron was determined with the light microscope at a 15003 final magnification. Any cell having a large nucleus with an identifiable nucleolus or nucleoli, showing Nissl substance and scattered basophilic granules throughout the cytoplasm was considered a neuron (Fig. 1). Because most glial cells, including oligodendrocytes and astrocytes, found in the AC rarely exceed 12 mm in their longest axis [13], an additional criterion to designate a neuron was that it should exceed 15 mm in the longest perikaryal axis as determined with the aid of a calibrated reticule adapted to one of the eyepieces (Zeiss, Germany). The studied sections were grouped into three commissural levels; namely, anterior, intermediate, and posterior. These levels correspond to the coronal planes 19, 20, and 21 described by Swanson [30]. Sectioning throughout the entire AC resulted in six sections per plane. The number of neurons

Fig. 1. Light micrograph from a coronal section through the caudal part of anterior commissure. Several interfascicular neurons (arrows) are seen among the commissural axons. Notice the presence of glial cells (arrowheads) alternating with interfascicular neurons. As shown both cell types are accommodated as rows of cells parallel to commissural axons. Bar510 mm. One-micrometer thin section. Toluidine-blue staining.

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per AC section was estimated by dividing the total number of neurons counted by 6—the number of sections.

2.2. Immunohistochemistry Four additional brains from adult animals were obtained following the procedure described for light and electron microscopy. The animals were perfused with 4% paraformaldehyde dissolved in phosphate–saline buffer, 0.1 M, pH 7.3. Then, each brain was removed and a tissue block containing the AC was cut with a razor blade and immersed into fresh cold (4 8C) fixative for 1 h. Afterwards, the tissue block was cryoprotected by leaving it overnight in 30% sucrose dissolved in PBS. Ten-micron thin sections were cut with a cryostat, following the horizontal plane. The resulting sections were incubated in anti-map-2 monoclonal antibody (MAB378, CHEMICON International, Temecula, CA, USA) (1:500), which stains dendritic trees and neuronal cell bodies [19]. Following overnight incubation with the primary antibody, immunoreactive sites were visualized by incubation with Cy-3conjugated goat anti-mouse antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) (1:500) for 3 h. Observations were made with a Nikon 6000 Eclipse microscope. More than 100 serial confocal images of the same field of stained tissue were taken at 0.2 mm focal plane intervals and then projected onto a single image.

Fig. 2. Incidence and distribution of interfascicular neurons throughout the rostro–caudal extent of the anterior commissure. Each drawing represents six collapsed coronal sections (i.e., one from every 20 mm) from the rostral (a) to caudal (c) anterior commissure. Each dot represents an interfascicular neuron with nucleolus. A caudal to rostral gradient of neuron incidence is evident; namely, 178 neurons in (a), 138 in (b), and 105 in (c). (Drawings modified from Swanson [30]).

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2.3. Rapid-Golgi impregnations Because a wide range of impregnation is obtained with the rapid-Golgi technique, 75 brains were processed. Male albino rats 25 days old (n535) or 12 weeks old (n540) were used. The brains from the young rats were mostly used to study short axon neurons and axonal inputs. Neuron size and dendritic characteristics were studied in the adult brain sections. Each of these types was determined according to the classical criteria described by ´ y Cajal [25] and Lorente de No´ [15]. Each rat was Ramon decapitated under deep anesthesia with pentobarbital (see above), and the brain was removed. A 3–4-mm thick slice containing the AC was obtained by means of two coronal sections. Each block was left for 5 days in a mixture containing 3% potassium dichromate and 0.25% osmium tetraoxide dissolved in deionized water and transferred to a 0.75% silver nitrate solution for 3 days. To obtain further detail about the pattern of axonal branching of AC axons and that of native commissural neurons, double and triple

Fig. 3. Survey confocal micrograph from a section incubated with an antibody to microtubule associated protein 2 (MAP 2). Three interfascicular neurons immunoreactive to MAP 2 are shown (white arrows). The radial disposition of primary dendrites is evident in the cell at the center. Notice the presence of numerous constrictions along the dendritic shafts. A dendrite (asterisk) proceeding from the septo–preoptic area projects to the domain of the posterior anterior commissure. Horizontal section. Black arrow5third ventricle.

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Fig. 4. High power confocal micrograph from an interfascicular neuron in the rostral anterior commissure. The tissue was incubated with a monoclonal antibody to microtubule associated protein 2 (MAP 2). Note the intense immunoreactivity in the dendritic processes and a weak labeling outlining the soma (arrow). Numerous varicocities are evident in the secondary and tertiary dendritic branches (arrowheads). Horizontal section.

impregnations [22] were also performed in 20 brains from the young animals. The resulting blocks were held with an external shell of paraffin and 100-mm thick sections were cut on a sliding microtome. The sections were made following coronal, sagittal, and horizontal planes. They were dehydrated in graded ethyl alcohols, cleared in terpineol–xylene, mounted, and cover-slipped. Neurons, axons, axonal collaterals, and putative terminals were drawn under a light microscope equipped with a camera lucida device. The length of the longest axis of the perikaryon was determined from drawings of 35 neurons obtained from adult rats. The drawings were digitized with a scanner adapted to a personal computer, and the axis was determined and measured with the assistance of the IPLab program. For illustration purposes, representative drawings were scanned, framed, and sized with the aid of photoediting software (AdobePhotoshop 5.5). The image shown in

Fig. 8a resulted from a series of 27 photographic exposures at different focal depths. The negatives from these images were scanned, and the areas with a sharp focus were ‘cut’, ‘pasted’, and ‘merged’ into one image with the photoediting software. No modifications other than contrast, size, and labeling were made to the original images.

3. Results

3.1. Light microscopy In 1-mm thin coronal sections stained with toluidineblue, most AC axons are found in compact bundles running horizontally and diagonally. Oligodendrocytes can be seen among these fascicles grouped in rows of 3–12 cells in parallel with the axon fascicles. Neurons are found

Fig. 5. Camera lucida drawing from an interfascicular neuron in the caudal anterior commissure. The axon (asterisk) leaves the soma at the base of a dendrite, projecting to the left parallel to a commissural axon (bottom). Primary dendrites leave the perikaryon in divergent directions generating successive secondary and tertiary branches. Note the beaded aspect of the dendritic processes. Few dendritic spines can be seen along the dendritic tree. Some commissural axons (soft pencil) give rise to short collaterals in apposition with both dendritic shafts and spines. Twelve-week-old rat; horizontal section; double silver impregnation. Fig. 6. Camera lucida drawing of a short axon interfascicular neuron in the rostral anterior commissure. Note the ascending axon (asterisk) that gives of several collaterals, which terminate as nest-like structures surrounding the profile of putative interfascicular neurons (arrows). A commissural collateral (arrowheads) descends and insinuates about on of these profiles. The cell contained by the box was successfully impregnated and is shown in detail in Fig. 10. Adult rat, coronal section, rapid-Golgi method. Calibration bar520 mm. Fig. 7. Camera lucida drawing from the rostral anterior commissure, depicting a projection interfascicular neuron. Most dendrites were omitted to illustrate the fork-like pattern of axonal bifurcation (asterisk). Note that as the axon ascends, it gives rise to collaterals that run laterally in opposite directions. Coronal section, adult rat brain. Rapid-Golgi method. Calibration bar530 mm.

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between the oligodendrocytes or near blood capillary vessels (Fig. 1). In adult animals, these oval or flameshaped neurons measure from 20 to 32 mm in the longest axis of the perikaryon. The nucleus is oval-shaped containing one or two nucleoli. The nuclear contour is pierced

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by one to three finger-like invaginations. Within the cytoplasm there are small clumps of Nissl substance, which may also be observed in proximal dendrites. These neurons consistently found among the axonal fascicles of the AC heretofore will be called interfascicular neurons

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Fig. 8. (a) Light micrograph of a projection interfascicular neuron. Notice that as the axon (arrow) ascends, it gives rise to paired horizontal collaterals that run horizontally, admixed with commissural axons (arrowheads). Triple impregnation, adult rat, caudal part of the anterior commissure. (b) Camera lucida drawing from the neuron illustrated in (a). Commissural fibers have been omitted to depict the fork-like pattern of ramification of the axon (ax). At the bottom a commissural axon collateral can be seen approaching the soma. Triple impregnation, adult rat, caudal part of the anterior commissure.

(IFN). The mean number of IFNs obtained from each 1-mm coronal section was: 29.3 (62.55) in the anterior, 23.3 (60.81) in the intermediate, and 17.3 (61.5) in the posterior sections (Fig. 2).

3.2. Immunohistochemistry With the confocal microscope, specimens incubated with MAP 2 antibody show immunoreactive neurons scattered throughout the AC (Fig. 3). The soma of most of these cells is weakly fluorescent, but dendrites display strong immunoreactivity, allowing identification of conspicuous varicosities along them (Figs. 3 and 4). These varicosities are more evident in the distal (i.e., tertiary)

Fig. 9. Camera lucida drawings illustrating the patterns of collateralization and termination of commissural axons. Fibers in (a) and (c) give rise to short transverse collaterals that shortly afterwards terminate as single boutons. The long collaterals (c) leave the anterior commissure, terminating upon neurons of the bed nucleus of the anterior commissure or septal and preoptic areas. Note that the fiber labeled (c) provides terminals en passage along the shaft (arrowheads). Fiber (d) originates short terminals (arrowheads) along its course. Fiber (e) is an example of bifurcating commissural axon. In (f), the interaction of commissural axons with the profile of interfascicular neurons and glia (soft pencil) is shown. Note that commissural axons give rise to short collaterals (arrowheads) that outline and appear to contact some of these cells. Coronal section. Twenty-five-day-old rat. Triple impregnation. Calibration bar530 mm.

branches. As a rule, the IFN dendrites remain within the AC. Other MAP 2-immunoreactive elements are dendritic processes projecting from neurons adjacent to the AC. These dendrites pierce the AC more frequently where it is adjacent to the bed nucleus of the AC, and rarely in the septo–preoptic area (Fig. 4).

3.3. Golgi-impregnated sections Because the structure of fiber collaterals and neurons appears to be similar throughout the AC, unless stated otherwise, the following description applies to both rostral and caudal AC. On the basis of the length and distribution of the axon, two types of IFNs are identified: short axon (i.e., internuncial) and projection (i.e., long axon). The perikaryon of the projection-type is characteristically oval or pear-shaped (Figs. 5 and 6) and averages 22 mm in the longest axis, whereas the soma of the internuncial type is stellate or rounded (Fig. 6) and ranges from 25 to 32 mm

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in the longest axis. The IFN dendritic tree consists of four or five proximal dendrites arranged in a radial pattern. Paired secondary branches emanate from these primary dendrites. A typical feature of the secondary dendrites is their beaded appearance, which becomes progressively more evident in the tertiary, usually terminal, dendrites. Horizontal sections reveal that most IFN dendrites run transversely or diagonally with respect to AC axons and assume a radial distribution (Figs. 5 and 6). Dendritic spines are moderately abundant, especially on secondary dendrites, and sparse on the soma or primary dendrites. Collaterals from commissural axons frequently contact the dendritic spines in what appear to be en passage or terminal boutons, respectively (see below). The short axon IFN sends a single axon shaft that soon branches into 2–5 collaterals. These collaterals give rise to thin varicose fibers that usually outline and appear to contact the neighboring IFNs’ somata (Fig. 6). In appropriate sections, terminals from short-axon IFNs may be seen surrounding the soma of long axon IFNs (Fig. 10). The projection or long axon IFN is characterized by an axon arising either from the soma (Fig. 5) or from a proximal dendrite (Figs 8a and b). The axon displays two distinct patterns. One consists of an axon that bends laterally and, after running a few hundred microns, divides into two or three collaterals that travel with commissural fibers (Fig. 5). The other type of axonal ramification consists of a series of successive branching that provide from 12 to 26 collaterals, which usually extend in opposite directions (Figs. 7, 8a and 8b). These collaterals run laterally admixed with the adjacent commissural fibers as far as 620 mm before abandoning the plane of the section. From the 23 neurons observed with this sort of fork-like axon, 20 were found within the medial third of the AC. Golgi-stained sections reveal that most commissural axons appear essentially unbranched throughout their course; however, some ramify at acute angles, generating thin collaterals that maintain the direction of the parent axon (Fig. 9e). Another ramification pattern of commissural axons consists of thin collaterals with varying lengths that leave the parent axon at a right angle. (Fig. 9a–d). These transverse collaterals approach adjacent IFN, possibly terminating on the spines or on the dendritic shafts (Figs. 5, 8b, and 10). In addition to transverse collaterals, IFNs may receive commissural inputs from axons passing adjacent to or through the rows of cells. These axons outline both glia and IFNs and issue short collaterals, probably terminating as en passage or terminal boutons (Fig. 9f).

3.4. Electron microscopy Interfascicular neurons are usually located near blood capillaries or oligodendrocytes (Fig. 11). Each IFN contains an oval nucleus displaying a distinct nucleolus surrounded by fine aggregates of heterochromatin. Even

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Fig. 10. Camera lucida drawing of an interfascicular neuron. Commissural axons running horizontally provide transverse collaterals that appear to contact the dendritic shafts (arrows) and dendritic spines (arrowheads). Note that an endogenous fiber (e) proceeding from a short axon neuron (shown in Fig. 6) appears to contact the neuron soma in a pericellular nest fashion. Coronal section from an adult rat brain. Rapid-Golgi method.

though the nuclear contour is generally smooth, it is nevertheless often pierced by one or two conspicuous invaginations from the nuclear envelope (Fig. 11). Occasional inclusions made up of fibril aggregates are seen in the nuclear matrix (Fig. 12) and are similar to the fibrillartype described in dorsal root ganglion cells [26]. The rough endoplasmic reticulum of these neurons adopts the form of short cisterns scattered throughout the perikaryon and proximal dendrites. Among the cisterns of the ergastoplasm and mitochondria, a few electron-dense, membranebounded structures can be identified (Fig. 12). In the juxtanuclear area, 2–4 Golgi complexes can be seen. Synaptic terminals may be found in apposition to either the soma (Fig. 12) or proximal dendrites. Synaptic boutons are occasionally observed among the neighboring commissural fibers. Finally, in the regions adjacent to some IFNs there are small areas of neuropil consisting of a few proximal dendrites, axo-shaft, axo-spinous synapses, terminal axons, and some myelinated axons (Fig. 11). Among these

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Fig. 11. Survey electron micrograph of an interfascicular neuron and adjacent neuropil. The neuron contains a lobulated nucleus (N) resulting from a conspicuous invagination. The surrounding perikaryon displays scattered mitochondria and abundant polyribosomes. Note the presence of several Golgi complexes within the yuxtanuclear area. The neighboring neuropil is composed of proximal dendrites (d) receiving synapses in their shafts (asterisks), and dendritic spines (s). The latter receive small synaptic boutons (arrows). Both neurons and neuropil are bounded by fascicles of commissural axons (Ax) and a part of an oligodendrocyte (Od).

neuronal elements (i.e., astro- and oligodendroglial), slender glial processes may also be encountered.

4. Discussion Commissural fiber systems are usually believed to be made up exclusively of axons conveying nerve impulses between equivalent forebrain areas. The present study documents and characterizes a set of distinct intracommissural neurons in the rat AC that because of their interaxonal location have been termed interfascicular neurons (IFN). The observation that IFNs share structural features with association and projection neurons suggests that in addition

to the role of interconnecting specific brain areas these AC neurons may also be a site where nerve impulses from commissural collaterals are processed. Silver-impregnated specimens suggest that IFNs receive inputs from specialized commissural efferents and from axons of the adjacent internuncial IFNs (Figs. 6, and 10). The pattern of transverse collateralization of commissural fibers has a striking resemblance to afferent fibers to the superior colliculus, spinal trigeminal nucleus, and some collaterals from the dorsal longitudinal fascicle to the oculomotor nuclei [25], all of which result from transverse branching of the afferent fiber. Along their course, commissural collaterals display en passage and terminal boutons on IFN dendrites, and this type of interaction

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Fig. 12. Electron micrograph depicting the cytoplasmic contents of an interfascicular neuron. At the upper left, part of the nucleus is shown. Within the karyoplasm there is a fibrillar inclusion (curved arrow). In the yuxtanuclear area, several Golgi complexes (G), electron-dense granules (asterisks), and mitochondria (m) can be seen. Throughout the periphery a multivesicular body (mv), polyribosomes, and rough endoplasmic reticulum (rer) are identified. Notice the presence of two synaptic boutons terminating on the soma (arrows).

[20,25] suggests that commissural collaterals terminate on IFNs, rather than simply passing through. Furthermore, information obtained by combined Golgi-high voltage electron microscopy has shown that the structures similar to those interpreted here as en passage and terminal boutons may also correspond to presynaptic elements [28]. The structure of short axon or internuncial IFNs meets the criteria of interneurons. An analogous mode of axon termination (i.e., nest-like structures) occurs in some short axon neurons of the primary acoustic nuclei [18], cerebellar cortex [25], and primary visual [31] and auditory [25] cortices. A subset of projection neurons is located primarily in the medial aspect of the AC. The axon of these cells divides dichotomously, resulting in a bilateral projection from each collateral. This axonal arrangement of IFNs resembles horizontal and biplume neurons of the isocortex [25] and represents a highly differentiated mode of IFN projection. The interaction between short axon and projection IFNs, coupled with the putative inputs provided by commissural collaterals, may constitute a basic four-element circuit within the AC. A model is proposed as a first approximation of this circuitry (Fig. 13). Nerve impulses issued by commissural collaterals arrive first. Second, commissural collaterals interact with the IFNs’ dendrites, an interaction

that is enhanced by the radial arrangement of the IFNs of the latter. Third, internuncial IFNs integrate commissural postsynaptic potentials, which, in the final step, control the discharge of projection IFNs. Such a circuit contains the essential elements of cylindrical physiological units in the mammalian cerebral cortex [16,17], which has received ample physiological validation [6,21]. Although much simpler than the organization of isocortical areas [15,31], the elements contained within the AC fulfill the basic substrata required for a process of neural modulation [18]. The brain areas influenced by the resulting nerve impulses generated by projection IFNs, deserve future investigation. Our observations suggest that the organization of the IFN is more complex than that originally surmised with respect to commissural neurons in the mose AC [29], fornix longus, and cerebellar white matter [25]. Rather than a one neuron relay system, the presence of short axon neurons in the AC is consistent with a putative presynaptic modulation of projection IFNs. The present findings challenge some of the basic and long-held beliefs concerning the anatomical and physiological partition of gray versus white matter. This opens the possibility that IFNs exist in other commissural and white matter projection systems and play a role in the modulation of nerve impulses transmitted by the adjacent axons.

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Fig. 13. Diagram illustrating a suggested mode of interaction among interfascicular neurons and commissural collaterals. Three neurons are shown, from left to right: short-axon (s), projection with a forked-type of axon (f), and unilateral projection (p). Commissural fibers (c) issue collaterals that terminate upon the three cell types (solid arrows). It is proposed that commissural collaterals control the discharge of short axon cells that, in turn, influence the activity of both types of projection cells (hollow arrows), resulting in a process of neuronal integration of commissural nerve impulses. See Section 4.

Acknowledgements The authors appreciate the useful suggestions of Drs. Larry W. Swanson and Carlos Valverde. We thank Dr Dorothy D. Pless for revising the grammatical aspects of ´ Viggiano for her the manuscript, Mrs. Carmen Mejıa technical help in processing tissues for immunohistochem´ istry, and Fis. Leopoldo Gonzalez Santos for editing the illustrations from confocal microscopy images. The grant ´ sponsor was Consejo Nacional de Ciencia y Tecnologıa, ´ Mexico (grant no. 28032-M) given to Dr. Jorge LarrivaSahd.

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