Robert S. Crissman

Organization of primary afferent axons in the trigeminal sensory root and tract of the rat

A combination of immunocytochemical and electron microscopic methods were employed to assess the organization of the trigeminal (V) spinal tract in adult rats. Immunostaining was employed at the light microscopic level to selectively label large myelinated (by using antibodies against neurofilament protein) and small unmyelinated (by using antibodies against calcitonin gene‐related peptide) primary afferents. In addition, the plant lectin Bandeiraea simplicifolia‐I was employed to histochemically label small unmyelinated primary afferents. Results from these experiments indicated that larger myelinated axons were distributed throughout the cross‐sectional extent of the V spinal tract (TrV), whereas smaller fibers were most numerous just below the pial surface. These results were confirmed with quantitative electron microscopy which demonstrated that the central portion of the V sensory root and TrV were composed primarily of larger myelinated fibers, whereas the periphery of the root and the portion of TrV just below the pial surface contained a higher percentage of smaller myelinated and unmyelinated axons. When considered together with results regarding the birthdates of neurochemically defined classes of V ganglion cells (White et al. [1994] J. Comp. Neurol. 350:397–411), these results suggest that TrV is laid down in a chronotopic fashion with the first axons forming its deeper portion and later arriving axons being added more superficially.

Development and plasticity of local intracortical projections within the vibrissae representation of the rat primary somatosensory cortex

Labelling with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (Di‐A) was used to assess the development of projections within the primary somatosensory cortex (SI) of rats aged between postnatal day 2 and 8 (P‐2 and P‐8). 1,1′‐Dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (Di‐I) was used in these same animals to label thalamocortical afferents. Particular attention was paid to the emergence of lamina IV intracortical projections that form a pattern complementary to vibrissae‐related thalamocortical afferents. A vibrissae‐related pattern of Di‐A‐labelled cells and fibers that was restricted largely to the septa regions was not apparent in rats killed on P‐2, but it was visible in animals killed on P‐4 and later ages. Tracing with biotinylated dextran amine (BDA) was used to assess intra‐SI projections of adult rats that sustained transection of the infraorbital nerve (ION) on P‐0 or P‐7 or implantation of a tetrodotoxin (TTX)‐impregnated polymer chip over the cortex on P‐0. Rats that sustained ION transection on P‐7 or that had TTX implants demonstrated normal patterns of projections within SI. The patterns of labelling in the supra‐ and infragranular layers of the cortices of the rats that sustained ION transection on P‐0 were generally similar to those in the other groups evaluated. However, in lamina IV, there was no organization that could be related to the distribution of the vibrissae. These results indicate that the vibrissae‐related pattern of intracortical projections within SI develops shortly after birth and that two manipulations that alter cortical activity, but not the patterning of thalamocortical afferents (application of TTX and transection of the ION after thalamocortical afferent patterns are established), have no significant effect on it. However, a manipulation that alters thalamocortical development (transection of the ION on P‐0) profoundly affects the patterning of intracortical connections.

Effect of neonatal axoplasmic transport attenuation in the infraorbital nerve on vibrissae-related patterns in the rat's brainstem, thalamus and cortex.

This study evaluated the effects of neonatal attenuation of axoplasmic transport in the infraorbital nerve (ION) on the organization of vibrissae‐related patterns in the rats CNS. Application of colchicine‐ or vinblastine impregnated implants to the ION from birth until postnatal day (P)6 to P10 resulted in a 92.4% reduction in the number of trigeminal (V) ganglion cells labelled by application of horseradish peroxidase to the vibrissa pad and a 44.8% decrease in the number of Nissl‐stained ganglion cells in the ophthalamic‐maxillary portion of the V ganglion. These implants also decreased the number of myelinated fibres in the ION. In normal rats killed on P6‐10, there was an average of 10 273±1259 myelinated axons in the nerve. In the animals with colchicine‐ or vinblastine‐treated implants, this value was 3891±1965. The highest axon count in an experimental animal was 9859. In all animals, axoplasmic transport attenuation resulted in the disappearance of normal vibrissae‐related cytochrome oxidase patterns in the brainstem, thalamus and primary somatosensory cortex. Axoplasmic transport attenuation did not result in the disappearance of vibrissae‐related ordering of V primary afferent terminal arbors, as demonstrated by anterograde labelling with neurobiotin. These results suggest that some factor conveyed from the periphery of the V ganglion and perhaps on to the brainstem is necessary for the maintenance of vibrissae‐related patterns in the thalamus and cortex.

Clorgyline treatment elevates cortical serotonin and temporarily disrupts the vibrissae-related pattern in rat somatosensory cortex.

Manipulation of cortical serotonin (5–HT) levels in perinatal rodents produces significant alterations in the development of the layer IV cortical representation of the mystacial vibrissae. Monoamine oxidase A (MAOA) knockout mice have highly elevated cortical 5–HT and completely lack barrels in somatosensory cortex (S–I). The present study was undertaken to determine whether the effects on thalamocortical development seen in MAOA knockout mice can be replicated in perinatal rats treated with an MAOA inhibitor and, second, to determine whether these effects persist with continued treatment or after discontinuation of the drug. Littermates were injected with either clorgyline (5 mg/kg) or sterile saline five times daily. Clorgyline administration from birth to postnatal day (P) 6, 8, or 10 produced increases of 1,589.4 ± 53.3%, 1660.2 ± 43.1% and 1,700.5 ± 84.5 %, respectively, in cortical 5–HT as compared with controls. Serotonin immunocytochemistry, 1,1`–dioctadecyl–3,3,3“,3`–tetramethylindocarbocyanine perchlorate (DiI) labeling of thalamocortical afferents and Nissl and cytochrome oxidase staining of layer IV cellular aggregates demonstrated that clorgyline treatment from P0 to P6 produced a complete absence of any segmentation of vibrissae–related patches in S–I. However, continued treatment until P8 or P10 did not prevent the appearance of these patches. Animals treated with clorgyline from birth to P6 and killed on P8 or P10 had increases of 546.8 ± 33.2% and 268.8 ± 6.3% in cortical 5–HT and they had qualitatively normal vibrissae–related patterns in S–I. These results indicate that clorgyline treatment produces a transient disruption of vibrissae–related patterns, despite the continued presence of elevated cortical 5–HT. J. Comp. Neurol. 427:139–149, 2000.

A Rapid Digestive Technique to Expose Networks of Vascular Elastic Fibers for Sem Observation

The NaOH sonication digestion technique permits rapid isolation and exposure of intact networks of elastic fibers in vascular tissue for 3-dimensional observation with the SEM. The configuration of the network of elastic fibers within the vascular wall of large elastic arteries (aorta) is generally agreed to be a flexible framework through which smooth muscle cells and collagenous fibers are interwoven. However, the configuration of elastic fiber networks in muscular arteries, medium sized veins and smaller vessels remains unknown. When the lengthy standard biochemical elastin purification techniques were applied to vessels containing lesser amounts of elastic tissue and finer elastic fibers, the vessels were completely digested. In contrast, the digestion and sonication technique isolated and exposed intact networks of delicate elastic fibers in blood vessels which do not contain large amounts of elastic tissue. Unfixed vessels were cut into short segments, placed in 0.5 N NaOH and sonicated for 20-40 min. The specimens were rinsed in deionized distilled H2O, then autoclaved for 30 min. The tissue was rinsed a second time, fixed and processed routinely for SEM. Elastic stains and enzymatic digestion with chromatographically purified elastase and collagenase confirmed that the digestion and sonication technique produced clean, isolated networks of elastic fibers. Knowledge of the configuration of the networks of elastic fibers in different vessels enhances understanding of distensibility characteristics of individual vessels and serves as a baseline for studying alterations in the elastic framework which occur during aging and disease processes such as atherosclerosis, arterial hypertension and aneurysms.

Numbers of Axons Innervating Mystacial Vibrissa Follicles in Newborn and Adult Rats

Electron-microscopic techniques were used to determine the numbers of axons in the deep vibrissal nerves innervating the C1 and C4 follicles in newborn and adult rats. All counts were made from thin sections taken after the nerve had entered the follicle capsule (FC). In newborn animals, the nerves supplying the C1 (n = 10) and C4 (n = 10) follicles contained an average (means +/- standard deviation) of 355.0 +/- 40.0 and 233.9 +/- 19.2 axons, respectively. In the adult animals (n = 10 for C1 and n = 9 for C4), the respective values were 314.4 +/- 26.2 and 233.3 +/- 34.4 axons. There were no significant differences between the values for the counts from the neonates and adults for either follicle (p greater than 0.01, independent t tests). In the vibrissal nerves of neonates, both degenerating axons and occasional growth cones were visible. Such profiles were not observed in the nerves taken from adults.

Reduction in the percentage of serotoninergic axons making synapses during the development of the superficial layers of the hamster's superior colliculus

Immunocytochemistry with an antibody directed against a serotonin (5-HT)-bovine serum albumin conjugate was combined with electron microscopy to assess the synaptic organization of the serotoninergic projection to the stratum griseum superficiale (SGS) of the superior colliculus of hamsters killed on postnatal days (P) 0, 3, 7, 12, 15, 20 and > 60. At least 200 5-HT-immunoreactive profiles were examined at each of these ages. In the newborn (P0) animals, 36.6% of the 5-HT-positive profiles made conventional synapses. This percentage remained fairly constant until P15 when only 17.3% of the profiles made conventional synaptic contacts. On P20, this value decreased to 10.5% and in the adult animals, it fell to 4.4%. These results thus indicate a dramatic age-related change in the synaptic organization of the 5-HT input to the hamsters superior colliculus.

Long-term effects of neonatal axoplasmic transport attenuation on the organization of the rat's trigeminal system

The current study examined the long‐term effects of infraorbital nerve (ION) axoplasmic transport attenuation with vinblastine on the organization of trigeminal (V) primary afferents and central vibrissae‐related patterns. Retrograde tracing and single unit recording were used to evaluate the innervation of vibrissae follicles in adult (P > 60) rats that sustained application of vinblastine to the ION at birth. Single units recorded from vinblastine‐treated animals yielded responses to deflection of a single vibrissa, and a significantly (P < 0.001) higher percentage of these cells (85.7%) showed rapidly adapting responses compared with normal rats (42.2%). Retrograde tracing revealed a qualitatively and normal distribution of V ganglion cells innervating A‐row and E‐row vibrissae follicles in vinblastine‐treated rats. Transganglionic tracing with horseradish peroxidase (HRP) demonstrated a qualitatively and quantitatively normal somatotopic organization of vibrissae follicle input to V nucleus principalis (PrV) and V subnucleus interpolaris (SpI) in the vinblastine‐treated animals. Despite the nearly normal mapping of V ganglion cell axons onto the vibrissae follicles and brainstem, staining for either cytochrome oxidase (CO) or parvalbumin failed to reveal vibrissae‐related patterns in PrV, SpI, or the magnocellular portion of V subnucleus caudalis in these animals. Labelling of thalamocortical afferents with HRP and staining for CO also failed to reveal a cortical vibrissae‐related pattern in the vinblastine‐treated rats. The present results indicate that although transient attenuation of axoplasmic transport with vinblastine has limited effects on the peripheral and central projections of surviving V primary afferents, it permanently disrupts the normal development and maintenance of central vibrissae‐related patterns. J. Comp. Neurol. 381:219‐229, 1997.

Effects of neonatal axoplasmic transport attenuation on the response properties of vibrissae-sensitive neurons in the trigeminal principal sensory nucleus of the rat

We have previously shown that attenuation of axoplasmic transport by application of vinblastine to the developing infraorbital nerve (ION) results in a loss of central vibrissae-related patterns that is not accompanied by changes in the receptive field sizes for the V primary afferents innervating the whisker follicles. The present study examines the relationship between the loss of central vibrissae-related patterns and alterations in the response properties of neurons in the V principal sensory nucleus (PrV) of adult rats that sustained application of vinblastine to the ION at birth. Absence of histochemically demonstrable vibrissae-related patterns in PrV resulted in only modest changes in the receptive fields and response properties of vibrissae-sensitive neurons in this nucleus that projected to the contralateral thalamus. Response latencies to electrical activation of the V ganglion were similar in treated and untreated animals. The mean receptive field size was significantly increased from 1.3 +/- 0.7 vibrissae in controls to 1.7 +/- 0.9 vibrissae in vinblastine-treated animals, and the percentage of cells yielding a tonic response to vibrissae deflection was markedly reduced (p < 0.01 for both measures). Phasically responding cells recorded in vinblastine-treated animals showed a significant reduction in the mean number of spikes per stimulus following deflection of the vibrissae in either the preferred or non-preferred direction relative to cells recorded in normal animals (p < 0.05). The present results indicate that disruption of the normal vibrissae-related aggregates of neurons in PrV by application of vinblastine to the ION has limited effects on the functional representation of the vibrissae in this nucleus.We have previously shown that attenuation of axoplasmic transport by application of vinblastine to the developing infraorbital nerve (ION) results in a loss of central vibrissae-related patterns that is not accompanied by changes in the receptive field sizes for the V primary afferents innervating the whisker follicles. The present study examines the relationship between the loss of central vibrissae-related patterns and alterations in the response properties of neurons in the V principal sensory nucleus (PrV) of adult rats that sustained application of vinblastine to the ION at birth. Absence of histochemically demonstrable vibrissae-related patterns in PrV resulted in only modest changes in the receptive fields and response properties of vibrissae-sensitive neurons in this nucleus that projected to the contralateral thalamus. Response latencies to electrical activation of the V ganglion were similar in treated and untreated animals. The mean receptive field size was significantly increased from 1.3 - 0.7 vibrissae in controls to 1.7 - 0.9 vibrissae in vinblastine-treated animals, and the percentage of cells yielding a tonic response to vibrissae deflection was markedly reduced (p < 0.01 for both measures). Phasically responding cells recorded in vinblastine-treated animals showed a significant reduction in the mean number of spikes per stimulus following deflection of the vibrissae in either the preferred or non-preferred direction relative to cells recorded in normal animals (p < 0.05). The present results indicate that disruption of the normal vibrissae-related aggregates of neurons in PrV by application of vinblastine to the ION has limited effects on the functional representation of the vibrissae in this nucleus.

The Three-Dimensional Configuration of the Elastic Fiber Network in Canine Saphenous Vein

Stereo scanning electron microscopy was used to study the normal arrangement of elastic fibers in canine saphenous veins. Selective alkali digestion and autoclaving exposed a network of interconnecting elastic fibers throughout the venous wall. 14 normal saphenous veins were surgically removed from 7 healthy dogs. Unfixed 1-cm segments of the isolated specimens were digested by sonication for 30 min in 0.5 N NaOH solution and then autoclaved for 30 min. The digested specimens were then fixed and routinely prepared for scanning electron microscopy. Large longitudinally oriented fibers in each tunica branched and anastomosed with adjacent fibers. Small fibers branched off at almost right angles and interconnected the longitudinal fibers. The configuration of the elastic network varied between the tunicae of the venous wall. The different networks were interconnected by anastomosing fibers of adjacent tunica to form a single elastic cylinder throughout the venous wall. The internal elastic lamina consisted of a single layer of fibers with a fishnet-like arrangement around the periphery of the luminal surface. Large, longitudinally oriented elastic fibers of the tunica media branched and anastomosed as in the internal elastic lamina. However, they also had tangential orientation and traversed different levels within the media. As a result, stratified layers were not formed. The external lamina consisted of several parallel layers of longitudinally oriented ribbons of elastic. Thick fibers interconnected these broad ribbons on the same and adjacent levels to form discontinuous layers. The configuration of the elastic network of the canine saphenous vein is compared to that previously reported for human saphenous vein. It is suggested that the architecture of this elastic network would contribute to vascular integrity and flexibility as well as aid in the distribution of stress throughout the venous wall. Basic knowledge of the elastic fiber network in this vein may be applied to future studies of diseased and grafted vessels.