Normal development and effects of neonatal infraorbital nerve damage upon the innervation of the trigeminal brainstem complex by primary afferent fibers containing calcitonin gene-related peptide.

Immunocytochemistry was used to study the normal development and response to infraorbital nerve (ION) damage of the innervation of the trigeminal (V) brainstem complex by axons recognized by an antibody directed against calcitonin gene-related peptide (CGRP). CGRP-like immunoreactivity (CGRPLI) was present in axons that occupied the outer V spinal tract (TrV) at all levels of the V brainstem complex. Almost no fibers terminated within V nucleus principalis (PrV), but there was dense CGRPLI in the supratrigeminal nucleus. There was also very little CGRPLI within rostral V subnucleus oralis (SpO). However, in the caudal one-half of the nucleus, a dense elongated patch of immunoreactivity was consistently present just medial to TrV. Only occasional CGRP-positive axons could be seen within V subnucleus interpolaris (SpI), but the paratrigeminal nucleus contained dense immunoreactivity. Trigeminal subnucleus caudalis (SpC) also contained CGRPLI that was very dense in lamina I and the outer portion of lamina II. Scattered terminals were also present in layers III and IV and dense terminal clusters were in lamina V. CGRP-immunoreactive neurons were present in the V ganglion by embryonic (E-) day 16 and immunoreactive axons could be seen in the V brainstem complex on E-17. At birth, CGRP-positive axons in the V brainstem complex had achieved a distribution very similar to that in adult rats. The major difference between the patterns of labelling in neonates and adults was the presence of relatively large numbers of CGRP-positive fibers in ventral PrV and SpO of the former animals. The disappearance of these fibers was completed by the middle of the third postnatal week. Transection of the ION on the day of birth had little effect upon CGRP in SpO, SpI, and SpC, but it did result in an increase in CGRP-positive fibers in PrV ipsilateral to the damaged nerve. When considered together with previous findings, these results suggest that CGRP-positive axons express this peptide well after they have entered the V brainstem complex and that the central terminal field of these fibers is not substantially altered by a manipulation which results in the death of nearly 60% of all V primary afferent neurons.

Effects of cortical and thalamic lesions upon primary afferent terminations, distributions of projection neurons, and the cytochrome oxidase pattern in the trigeminal brainstem complex.

Early postnatal lesions of the primary somatosensory cortex alter the vibrissa-related cytochrome oxidase (CO) pattern in nucleus principalis (PrV) of the rat's trigeminal (V) brainstem complex (Erzurumlu and Ebner, '88: Dev. Brain Res. 44:302-308). At present, the reason for this change is not clear. It may be that the corticotrigeminal projection is necessary for the maintenance of vibrissa-related patterns in PrV. However, it is also possible that the loss of the normal pattern of CO activity reflects a change in the organization of brainstem cells resulting from transneuronal retrograde degeneration. In order to address this question, we made lesions of either the primary somatosensory cortex (S-I) or ventrobasal thalamus (VB) in newborn rats and directly assayed distribution of V primary afferents by transganglionic transport of horseradish peroxidase and V-thalamic neurons by retrograde transport of either fluorogold or true blue. Neonatal cortical and thalamic lesions produced no qualitative change in the distribution of primary afferent terminals in either PrV or V subnucleus interpolaris (SpI) beyond that which could be attributed to shrinkage of the brainstem resulting from retrograde degeneration. Most importantly, the "patchy" pattern of terminations observed in normal rats remained apparent in the brain-damaged animals. The normal distribution of V-thalamic neurons in PrV was disrupted by both cortical and thalamic lesions. These cells are normally patterned in a way that matches the distribution of primary afferent terminals and thus that of the mystacial vibrissae. This was not the case in the neonatally brain-damaged rats. Taken together, these results are consistent with the conclusion that neonatal cortical and thalamic lesions disrupt the normal CO pattern in PrV primarily because of their effects upon the patterning of brainstem cells. The present findings demonstrate further that clustering of primary afferents does not require a normal complement of postsynaptic neurons.

Morphological characteristics of low-threshold primary afferents in the trigeminal subnuclei interpolaris and caudalis (the medullary dorsal horn) of the golden hamster.

Intra-axonal recording and horseradish peroxidase (HRP) injection techniques were employed to define the response characteristics of low-threshold, rapidly conducting trigeminal primary afferents and the morphological features of their axon arbors in subnucleus interpolaris and subnucleus caudalis (or the medullary dorsal horn; these last two terms are used synonomously throughout the paper). A total of 61 such afferents were characterized and recovered. Of these, ten gave rapidly adapting (RA) and 17 slowly adapting (SA type I) responses to vibrissa deflection. Twenty were sensitive to guard hair deflection and 14 were responsive to indentation of the hairy skin. The vibrissa-sensitive primary afferents were all quite similar morphologically. Primary collaterals proceeded directly, in a radial fashion, to their zone of termination and gave rise to dense and compact arbors. These tended to be larger in the medullary dorsal horn (MDH) than in interpolaris and they also gave rise to more boutons in the former nucleus. Guard hair afferents generally had smaller arbors and gave rise to fewer boutons than vibrissa-sensitive axons. Like vibrissa afferents, their arbor were generally circumscribed in both interpolaris and MDH, but they were larger in the latter nucleus. Skin-sensitive afferents had arbors that tended to be somewhat larger than those of vibrissa- or guard-hair-related fibers. Unlike the other fiber types, the arbors of skin-sensitive afferents were on average larger in interpolaris than MDH. Quantitative analysis of the morphological data from well-filled examples from each of these four functional types verified our qualitative impressions regarding differences between interpolaris and MDH collaterals of a given fiber-type. Statistical comparison of data from different functional classes indicated trends that supported our qualitative impressions, but none of these was statistically significant. The topography of the trigeminal primary afferent input to interpolaris was organized such that the head was inverted and fibers with caudal receptive fields terminated in the lateral portion of the nucleus. This was true for all of the functional afferent types that we examined. Vibrissa-related fibers differed from nonvibrissa afferents in that they tended to avoid the most rostral portion of interpolaris. In the MDH, the primary afferent representation of the head was also inverted, but fibers with caudal facial receptive fields tended to terminate medially rather than laterally.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for prenatal competition among the central arbors of trigeminal primary afferent neurons: single axon analysis.

Previous studies from this laboratory have demonstrated that prenatal damage to vibrissae follicles results in significant increases in the brainstem representations of the remaining vibrissae as demonstrated by staining for the mitochondrial enzyme cytochrome oxidase (CO). Because CO is primarily a postsynaptic marker, these results do not directly address the question of whether there were changes in the projections of primary afferent fibers. To address this issue, we made intra-axonal recordings from individual vibrissa-related primary afferents in rats that sustained damage to vibrissae follicles on embryonic day 17, and then injected horseradish peroxidase (HRP) into these axons to visualize their terminal arbors in the brainstem at the level of trigeminal subnucleus interpolaris (SpI). All vibrissae-related primary afferents responded to deflection of one and only one vibrissa, and the terminal arbors of axons (N = 47) recovered from animals that sustained fetal peripheral lesions were significantly larger than those (N = 23) from normal rats. Fibers from fetally damaged animals had increased total fiber lengths and numbers of branch points. These results indicate that reduced competition among primary afferent axons results in increases in the terminal arbors that remain. These increases occur without any significant alteration in their peripheral receptive fields.

Intracellular recording and injection study of corticospinal neurons in the rat somatosensory cortex: effect of prenatal exposure to ethanol.

The effects of prenatal exposure to ethanol on the structure and function of corticospinal neurons was investigated. The subjects were the 3-4-month-old offspring of hooded rats fed a nutritionally balanced liquid diet containing 6.7% (v/v) ethanol (Et), pair-fed a nutritionally matched isocaloric diet (Ct), or fed chow and water (Ch). Corticospinal neurons in primary somatosensory cortex were examined by intracellularly recording and filling cells that were driven by antidromic stimulation of the pyramidal decussation. In the control rats, corticospinal neurons comprised a homogeneous morphophysiological population. Morphologically, all of the antidromically driven cells examined were pyramidal neurons with cell bodies in layer Vb. The dendrites of these neurons were spinous and branched within layers I, IV, and V. Their axons arborized within layers IV, V, and VI and some collaterals extended laterally for distances up to 2.6 mm from the cell body. The mean conduction latency was 3.6 and 3.4 msec for Ch- and Ct-treated rats, respectively. In Et-treated rats, corticospinal neurons constituted a heterogeneous population. The laminar distribution of the corticospinal neurons in Et-treated rats was broad; the cell bodies of labeled neurons were in layers II, IV, V, and VI. The dendrites of layer Vb neurons were spinous; however, many of the spines appeared dysmorphic and the density of spines was significantly greater (32%) in Et-treated rats than in Ct-treated rats. Although the dendritic branching pattern for layer Vb neurons was similar to that described for the controls, a Sholl analysis showed that the complexity and extent of their dendritic trees were significantly greater in Et-treated rats. The axons of all layer Vb neurons in Et-treated rats had long horizontal processes that arborized in layers IV-VI, and some neurons also had an array of collaterals that ascended to layer I. The mean conduction latency for layer Vb neurons was 3.9 msec. The structure and function of ectopic neurons (those in layers II, IV, Va, Vc, and VI) in Et-treated rats differed markedly from those of the layer Vb neurons. Morphologically, the dendritic and axonal fields of these neurons were narrower than for the layer Vb neurons, and the ectopic neurons had a mean conduction latency of 7.1 msec. The heterogeneity of the population of corticospinal neurons in Et-treated rats may result from the effects of ethanol on early events in neuronal development such as neuronal generation and migration.

Patterning of the neocortical projections from the raphe nuclei in perinatal rats: investigation of potential organizational mechanisms.

Serotoninergic (5-HT) fibers in the cerebral cortex of perinatal rats have a pattern that coincides with the boundaries of primary sensory areas and within the primary somatosensory cortex form the rattunculus. This patterned immunoreactivity (IR) appears about 60 hours after birth and disappears between postnatal days (P-) 12 and 15. Three experiments were carried out to evaluate mechanisms that might underlie the precise patterning of the 5-HT-IR. Retrograde labelling with fluorescent tracers in perinatal rats revealed only a coarse rostrocaudal topography in the raphe-cortical projection and the existence of raphe cells projecting to multiple cortical locations. Thus, a precise point-to-point, raphe-cortical projection does not underlie the patterned cortical 5-HT-IR. Ablation of the thalamus prior to the age at which patterned 5-HT-IR could be seen in the developing cortex caused a complete loss of patterned immunoreactivity. This suggests that 5-HT fibers may require the presence of thalamocortical axons to achieve the pattern observed in normal animals. Serotoninergic raphe neurons transplanted to the cortices of newborn rats exhibited extensive axonal outgrowth, but did not form a somatotopic pattern. This result also suggests that specific spatiotemporal interactions between growing 5-HT and thalamocortical axons may be necessary for the somatotopic patterning of the former fibers.

Thalamic processing of vibrissal information in the rat. I. Afferent input to the medial ventral posterior and posterior nuclei.

Retrograde tracing with true blue (TB) and diamidino yellow (DY) and anterograde tracing with either wheatgerm agglutinin-conjugated horseradish peroxidase (WGA-HRP) or Phaseolus vulgaris leucoagglutinin (PHA-L) were employed to investigate the projections from trigeminal nucleus principalis (PrV) and trigeminal subnucleus interpolaris (SpI) to their targets in the medial ventral posterior (VPM) and posterior (POm) nuclei of the thalamus. Many more cells in both PrV and SpI were labeled by tracer injections into VPM than into POm. Only a very small number of double-labeled neurons were observed in either PrV or SpI. However, a significantly higher percentage of SpI cells projected to POm or to both POm and VPM than was the case for PrV. Anterograde tracing with WGA-HRP showed that the projections from both PrV and SpI to VPM were much denser than those from the same nuclei to POm. Small injections of PHA-L into either PrV or SpI produced a focus of fairly dense labeling in VPM and much more diffuse terminal labeling in POm. These anatomical data provide evidence for two separate trigeminothalamic pathways, one originating from PrV and the second originating from SpI. Both of these pathways converge and diverge at the thalamic level. That is, information from the PrV pathway and from the SpI pathway are both provided to VPM in a morphologically restricted fashion and to POm in a morphologically widespread fashion.

Thalamic processing of vibrissal information in the rat: II. Morphological and functional properties of medial ventral posterior nucleus and posterior nucleus neurons.

Extracellular recording, intracellular recording, intracellular horseradish peroxidase injection, and receptive field mapping techniques were employed to evaluate the physiological and morphological properties of medial ventral posterior nucleus (VPM) and posterior nucleus (POm) neurons in normal adult rats. Overall, we physiologically characterized 148 VPM and 121 POm neurons. Over 82% of the VPM cells were excited only by deflection of one or more mystacial vibrissae, 10% were activated by displacement of guard hairs, and the remainder were either excited by indentation of the skin or were unresponsive. Less than 40% of the POm cells were activated by vibrissa deflection, 18% were excited by displacement of guard hairs, and another 17% were unresponsive. Most of the rest of the POm cells were excited by stimulation of skin, mucosa, or activation of muscle-related afferents. Small percentages of POm cells responded only to noxious stimulation, were classified as having a wide dynamic range, or were inhibited by peripheral stimulation. Electrical stimulation of either PrV or SpI activated most neurons in both VPM and POm. This excitation was almost invariably followed by a long-lasting hyperpolarization which was generally strong enough to prevent responses to either electrical stimuli delivered in the brainstem or mechanical stimulation of the periphery. The receptive fields of vibrissa-sensitive cells in POm were generally much larger than those of cells in VPM. Data obtained with extracellular recording indicated that VPM and POm cells responded to an average of 1.4 and 4.0 vibrissae, respectively. Intracellular recording from smaller samples of VPM and POm cells demonstrated the existence of inputs that were insufficient to produce spikes from the cell, but did yield epsp's. When both sub- and suprathreshold excitation were considered, the average number of vibrissa in the receptive field of a VPM cell was 2.7 and the value for POm cells became 7.8. HRP-filled neurons recovered in POm (N = 20) generally had much larger dendritic arbors than neurons in VPM (N = 31). For the former cells, the size of the dendritic tree was significantly correlated with the number of vibrissa to which the cell responded; for the latter neurons, it was not.

Cholecystokinin concentrations and peptide immunoreactivity in the intact and deafferented medullary dorsal horn of the rat.

To further address the hypothesis that cholecystokinin (CCK) in the medullary dorsal horn (MDH) arises from intrinsic or higher-order neurons, CCK-8-specific radioimmunoassay (RIA) and immunohistochemical (IHC) experiments were carried out in adult rats after trigeminal tractotomy. RIA of punches from deafferented superficial layers of the MDH revealed no significant change in CCK levels vs. the control right side. In this same area, IHC revealed modest reductions in CCK, gastrin, and substance P staining. Calcitonin gene-related peptide (CGRP) staining was reduced substantially. Gastrin immunoreactive cell bodies, present normally in inner lamina II, were reduced in number. RIA and IHC methods were also used to assess MDH CCK concentrations in adult rats subjected to left infraorbital nerve section at birth. The left medulla contained significantly higher levels of CCK than the control right medulla (1.27 +/- 0.19 vs. 0.97 +/- 0.11 ng/mg protein). IHC revealed a dense band of CCK-like staining in laminae I and II ipsi- and contralateral to the lesion. Thus, neonatal deafferentation elevates medullary CCK. To determine if the neonatal lesion-induced increase in medullary CCK is due to primary afferent or higher-order reorganization, RIA and IHC experiments were run after infraorbital nerve section at birth and trigeminal tractotomy in adulthood. RIA revealed no significant change in CCK levels caudal to the tractotomy, although they were higher than control levels in 9 of 12 cases. IHC revealed modest reductions in CCK, substance P, and gastrin staining that resembled the reductions observed in tractotomy-alone cases. These data suggest that 1) most MDH CCK is of non-primary afferent origin, 2) gastrin immunoreactivity in layer II probably originates in CCK-containing cells intrinsic to layer II, the expression of which is dependent upon trigeminal primary afferent input, 3) neonatal V deafferentation induces increased CCK in the superficial MDH, reflecting reorganized intrinsic or higher-order inputs, and 4) higher-order substance P in the MDH is robust.

Patterning of local intracortical projections within the vibrissae representation of rat primary somatosensory cortex.

Anterograde and retrograde tracing with biotinylated dextran amine and Phaseolus vulgaris leukoagglutinin was used to assess projection patterns within the vibrissae representation of the rat's primary somatosensory cortex (S-I). Large and small injections of either tracer into the center of the vibrissae representation yielded dense anterograde and retrograde labelling throughout much of the tangential extent of the vibrissae representation within S-I. In all layers, the pattern and extent of retrograde and anterograde label was in rough congruence. The organization of this labelling varied across cortical layers. In layers II and III, labelled fibers extended away from injection sites in all directions and yielded a uniform pattern, which decreased in density with increasing distance from the tracer injection. There was a tendency for labelling to be more extensive along the representation of the row of vibrissae follicles that included the injection site than across rows. There was also a tendency for anterograde labelling to be more extensive in the direction of the representation of follicles more rostral on the face than that injected. In lamina IV, both labelled fibers and cells were restricted for the most part to the septa regions between the barrels. However, a small number of retrogradely labelled neurons were also located in the barrels (approximately one-ninth of the number found in the septa). The pattern observed in laminae II-III was repeated in layers V and VI. In these laminae, there was no evidence of a pattern of intracortical connections related to the vibrissae representation in overlying lamina IV.

Birth dates and survival after axotomy of neurochemically defined subsets of trigeminal ganglion cells.

Trigeminal (V) ganglion cells with different neurochemical phenotypes or different birth dates are affected differently by neonatal axonal transection. The aim of the present study was to determine if V ganglion cell birth date and neurochemical phenotype were correlated and if these two variables could be related to responses to neonatal axonal transection. Immunocytochemistry, histochemistry, and [3H]thymidine labelling were used to determine the birth dates of V ganglion cells recognized by antibodies directed against neurofilament protein (NF), calcitonin gene-related peptide (CGRP), and substance P (SP) and those that bound the lectin Bandierea simplicifolia-I (BS-I). All V ganglion cells were born between embryonic days (E-) 9.5 and 14.5. All ganglion cells were born between E-9.5 and E-14.5. In a normalized population (percentages normalized to equal 100%), over 90% of NF-positive V ganglion cells were born between E-10.5 and E-12.5. The majority of CGRP-positive and SP-positive ganglion cells (> 90%) were generated from E-13.5 to E-14.5 and E-12.5 through E-14.5, respectively. Almost 85% of BS-I-positive ganglion cells were generated on E-12.5 through E-14.5. Previous results and additional data from this study indicated that NF- and BS-I-positive ganglion cells are proportionally more likely to be lost after neonatal axotomy and that SP-positive cells are more likely to remain. The percentage of CGRP-positive cells in the V ganglion was not significantly altered by neonatal infraorbital nerve transection. Overall, these findings do not indicate a strong relationship between cell birth date and the probability of survival after neonatal axonal damage for all V ganglion cell phenotypes.

Organization of the projections from the trigeminal brainstem complex to the superior colliculus in the rat and hamster: anterograde tracing with Phaseolus vulgaris leucoagglutinin and intra-axonal injection.

Anterograde tracing with Phaseolus vulgaris leucoagglutinin (PHA-L) and intra-axonal recording and injection techniques were employed to describe the projection from the trigeminal (V) brainstem complex to the deep laminae of the superior colliculus (SC) in the hamster and the rat. The organization of these projections was the same in the two species. Deposits of PHA-L into V nucleus principalis (PrV) produced labelled axons and boutonlike swellings in the lower stratum griseum intermediale (SGI) and upper stratum album intermedium (SAI) in the SC bilaterally. Plots of boutonlike swellings indicated that the terminals of this projection were arrayed in clusters. Nucleus principalis also projected to the stratum griseum profundum (SGP) and stratum album profundum (SAP). This deeper projection did not terminate in clusters and it was most prominent in the lateral SC. The ipsilateral PrV-SC projection appeared to arise mainly from axons that recrossed the midline at the level of the SC commissure. Reconstruction of individual PHA-L labelled fibers demonstrated that single axons gave rise to terminals on both sides of the midline. Deposits of PHA-L into V subnucleus interpolaris (SpI) yielded results that were identical to those obtained with PrV injections with one exception: none of these deposits produced any labelled terminals in the ipsilateral SC. Deposits of PHA-L into V subnucleus caudalis (SpC) produced only sparse labelling in SC. Most labelled swellings were located in the SGP and SAP and they were visible only in the SC contralateral to the PHA-L injection site. Single axons arising from cells in SpI were recorded and injected with horseradish peroxidase (HRP) in the hamster's SC. These fibers all responded to stimulation of multiple mystacial vibrissae and gave rise to 2-5 clusters of bouton-like swellings in the lower SGI and upper SAI.

Parvalbumin and calbindin immunocytochemistry reveal functionally distinct cell groups and vibrissa-related patterns in the trigeminal brainstem complex of the adult rat.

Immunocytochemistry for calbindin (CA) and parvalbumin (PA) was combined with retrograde tracing from the thalamus, superior colliculus (SC), and cerebellum to define the ascending projections of neurons in the rat's trigeminal (V) brainstem complex that express immunoreactivity for these calcium binding proteins. Many PA-immunoreactive neurons were observed in trigeminal nucleus principalis (PrV). Many of these cells projected to thalamus and a few sent axons to SC. In ventral PrV, PA-immunoreactive neurons were arranged in a vibrissa-related pattern. A very small number of large CA-immunoreactive neurons were observed in dorsomedial PrV. None of these cells were labeled by our tracer deposits. Small neurons in V subnucleus oralis (SpO) were also immunoreactive for PA, but none were retrogradely labeled. A small percentage of the large neurons in SpO were CA-immunoreactive; many of these were retrogradely labeled by tracer injections in the thalamus and/or SC. In V subnucleus interpolaris (SpI), many small to medium sized cells were PA-positive and they were arrayed in a vibrissae-like pattern. None of these neurons were retrogradely labeled from any of the above-listed targets, but many were retrogradely labeled by tracer injections into ipsilateral PrV. SpI also contained many large CA-immunoreactive cells. Many of these projected to the thalamus and/or SC and some were also retrogradely labeled by tracer injections into ipsilateral PrV. In V subnucleus caudalis (SpC), very dark PA-immunoreactive neurons were located in the inner part of lamina II and less often in laminae I. Lightly labeled cells were located in the magnocellular laminae and formed vibrissa-related aggregates. None of these neurons were retrogradely labeled by our tracer injections. CA-immunoreactive cells were located throughout the depth of lamina II in SpC and smaller numbers were also visible in lamina I and layers III-V. A small percentage of the CA-positive cells in lamina I and in the magnocellular layers were retrogradely labeled from the thalamus. These data indicate that PA and CA antisera identify two cell populations in whisker-related regions of the V brainstem complex and that PA cells are somatotopically patterned in PrV, SpI, and SpC. These markers also distinguish two cell groups in superficial laminae of the medullary dorsal horn.

Neonatal infraorbital nerve transection in rat results in peripheral trigeminal sprouting.

Retrograde tracing techniques were employed to determine whether transection of the infraorbital (IO) nerve in either newborn or adult rats resulted in peripheral sprouting by undamaged trigeminal (V) axons. The IO nerve was sectioned just behind the vibrissa pad, either on the day of birth or when animals reached at least 60 days of age. After an additional 60 days, the same nerve was retransected in the orbit; horseradish peroxidase (HRP) or diamidino yellow (DY) was injected into the central portion of the vibrissa pad; and animals were killed 2-3 days later. In the neonatally nerve-damaged rats, this procedure invariably labelled primary afferent neurons in both the ipsilateral and contralateral V ganglia. On the ipsilateral side, these cells were located in the caudal portion of the ophthalmic-maxillary region and, less often, in the mandibular division. Their average diameter was 22.6 micron (s.d. = 5.6). On the contralateral side, most labelled ganglion cells were visible in the anteromedial part of the ophthalmic-maxillary region but a few could also be seen in the mandibular division. Their average diameter was 21.1 micron (s.d. = 5.5). No labelled ganglion cells were observed in adult rats subjected to the same series of manipulations. In a separate series of neonatally nerve-damaged animals, the above-described procedures were combined with neonatal injection of capsaicin in an effort to determine whether the observed sprouting was dependent upon the presence of large numbers of unmyelinated axons. The addition of this treatment reduced the number of labelled cells in both the ipsilateral and contralateral ganglia, but it did not alter either their distribution or average soma diameter. In a final experiment, sequential double-labelling techniques were used to determine whether the V axons that projected to the vibrissa pad via non-IO nerve branches were the result of sprouting by undamaged ganglion cells or arose from neurons that had originally projected into the IO nerve, were axotomized by our lesions, and regenerated to the vibrissa pad via another V branch. Here, the long-lived retrograde tracer true blue (TB) was injected into the vibrissa pad 6-8 hours before the neonatal nerve cut and DY was deposited into the pad after transection of the regenerate IO nerve in adulthood. Double-labelled cells in this experiment would have projected to the vibrissa pad via the IO nerve at birth and regenerated to it via another V branch in adulthood. Nearly 55% of the DY-labelled cells in this experiment also contained TB.(ABSTRACT TRUNCATED AT 400 WORDS)

Laminar and areal differences in the origin of the subcortical projection neurons of the rat somatosensory cortex.

Fluorescent retrograde tracing techniques were employed in a double-labelling paradigm to determine the distribution of corticospinal, corticotectal, and corticotrigeminal projection neurons in layer Vb of the adult and neonatal rat somatosensory cortex. The double-labelling paradigm allowed a direct comparison of the cortical distribution of neurons projecting to each target and identification of neurons projecting to more than one target. In the adult rat, each population of projection neurons was found to have a unique laminar and/or areal distribution. Corticospinal projection neurons were located throughout the width of layer Vb in the medial granular portion of somatosensory cortex, while corticotrigeminal projection neurons were distributed throughout the width of layer Vb in the more laterally located dysgranular portion of somatosensory cortex. Corticotectal projection neurons were located more superficially in layer Vb than either corticospinal or corticotrigeminal projection neurons and found scattered throughout both dysgranular and granular somatosensory cortex. Each combination of subcortical injections also resulted in double labelling a small percentage of uniquely distributed neurons. These distribution differences coupled with measurements of cell size allowed us to identify the parent population of the dual projection neurons. Subpopulations of corticotectal neurons also project to the brainstem trigeminal complex and to the spinal cord. Subpopulations of corticotrigeminal neurons also project to the spinal cord, and a proportion of corticotrigeminal neurons projects to at least two targets within the brainstem trigeminal complex (nucleus principalis and subnucleus interpolaris). In the adult rat, corticospinal neurons (as defined by either laminar position or somal size) did not appear to give off collaterals to either the superior colliculus or brainstem trigeminal complex. In the neonatal rat, double-labelled neurons which project to both the spinal cord and the tectum are distributed throughout the full width of layer Vb, rather than restricted to the superficial portion of the layer as in the adult rat. Further, it appears as if the ontogenetic change in the laminar distribution of corticospinal and tectal projection neurons is achieved by mechanisms of selective process elimination rather than cell death. These results are discussed in terms of both the developmental factor which may contribute to the discrete distribution of cortical projection neurons found in the adult and the functional significance of bifurcating projection neurons.

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.

Sensitive period for lesion-induced reorganization of intracortical projections within the vibrissae representation of rat's primary somatosensory cortex.

Previous experiments from this laboratory demonstrated that intracortical connections in lamina IV of the rat primary somatosensory cortex (SI) are most dense outside the patches of cytochrome oxidase (CO) staining that correspond to the mystacial vibrissae. This pattern of intracortical connections becomes apparent on postnatal day 4 (P-4), at least 2 days after the appearance of the vibrissae-related pattern of thalamocortical afferents. Transection of the infraorbital nerve (ION) on the day of birth (P-0) disrupts both the CO and intracortical projection patterns. This series of experiments was undertaken to determine whether the patterning of either thalamocortical afferents or intracortical projections defines the end of the period over which peripheral damage can alter intracortical projections in lamina IV of SI. The infraorbital nerve (ION) was transected in different cohorts of rats on P-1 through P-5, and animals were allowed to survive > or =45 days, at which time biotinylated dextran amine (BDA) injections were made into the SI. After 7 days, animals were killed, and alternate cortical sections were processed for the demonstration of BDA or CO. Transection of the ION on P-1 or P-2 altered the patterning of both CO and intracortical connections in the SI. In contrast, cutting the ION on P-3 left the pattern of CO densities in the SI intact, but significantly altered the patterning of intracortical connections. Transection of the nerve on P-5 resulted in qualitatively and quantitatively normal patterns of both CO densities and BDA-labelled intracortical projections. These results indicate that the establishment of a stable barrel pattern in layer IV of the SI is not sufficient for normal adult patterning of intracortical projections in this lamina. However, once the mature pattern of intracortical projections in layer IV is established, ION lesions can no longer alter it.

Effect of activity blockade on changes in vibrissae-related patterns in the rat's primary somatosensory cortex induced by serotonin depletion.

Depletion of cortical serotonin (5-HT) during development results in a decrease in the size of the patches of thalamocortical afferents representing the mystacial vibrissae in lamina IV of the primary somatosensory cortex (SI). We previously suggested that this change may be due to a reduction in 5-HT-induced suppression of thalamocortical activity in these animals. The present experiments directly tested the role that modulation of activity may play in the morphologic changes observed after reducing cortical 5-HT concentrations. Serotonin was depleted from the cortex by systemic administration of 5,7-dihydroxytryptamine (5,7-DHT, 100 mg/kg) on the day of birth in animals that also had either tetrodotoxin (TTX)-impregnated or control implants placed unilaterally over the developing SI on this day. Other rat pups were treated with TTX-impregnated or control implants alone. Administration of 5,7-DHT reduced cortical serotonin levels and this effect was not significantly modified by the presence of either control or TTX-impregnated cortical implants. Administration of 5,7-DHT reduced the cross-sectional area of the cortical patches, demonstrated by acetylcholinesterase, corresponding to the vibrissae by 19.9% (P < 0.05). A similar reduction was observed in the animals treated with both 5,7-DHT and TTX-impregnated implants. Treatment with TTX-impregnated implants alone resulted in a 3.1% increase in patch size (P > 0.05). None of the treatments significantly altered the overall area of the part of SI devoted to the representation of the long mystacial vibrissae. These results suggest that the effects of 5-HT depletion on the size of the cortical patches representing the long vibrissae are independent of activity that can be blocked by administration of TTX.

Organization and actions of the noradrenergic input to the hamster's superior colliculus.

Immunocytochemistry using antisera directed against dopamine-beta-hydroxylase (DBH) was used to determine the organization of the noradrenergic (NE) input to the hamster's superior colliculus (SC). Immunocytochemistry for DBH was combined with retrograde transport of fluorogold (FG) to determine the sources of NE input to SC. Microiontophoretic techniques were used together with extracellular single unit recording and receptive field mapping techniques to determine the manner in which NE influenced the responses of individual SC neurons. The hamster's SC contained numerous DBH-positive fibers but no immunopositive cells. These fibers formed a plexus that was most dense in the lower stratum griseum superficiale (SGS). The density of DBH-positive fibers was very low in the stratum opticum (SO) and increased in density in the stratum griseum intermediale (SGI) and the other deep layers. When FG injections into the SC were combined with immunocytochemical detection of DBH, double-labeled cells were observed in the contralateral locus ceruleus. DBH-positive neurons were observed in several other portions of the mesencephalon and pons, but none of these were labelled with FG. The effects of NE iontophoresis were assessed for a total of 135 SC neurons. In 74% (N = 100), NE reduced spontaneous and/or stimulus evoked activity. In 3% (N = 4 cells), NE increased activity, and in 23% (N = 31 cells) it had no effect. These percentages were essentially the same for superficial layer visual cells and somatosensory neurons in the deep laminae. The effect of NE iontophoresis upon signal to noise ratios was assessed for 46 visual and 56 somatosensory neurons. For 54% (N = 25) of the visual cells and 16% (N = 9) of the somatosensory cells, NE iontophoresis decreased signal to noise ratios. For 13% (N = 6) of the visual cells and 21% (N = 12) of the somatosensory cells, NE iontophoresis increased signal to noise ratios. The effects of NE on the responsivity of SC neurons were antagonized by propranolol (86% of the 21 cells tested), sotalol (67% of the six cells tested), and atenolol (effective in the single cell tested). All these agents are beta-adrenergic antagonists. The single alpha-adrenergic antagonist that we evaluated, corynanthine, potentiated the effects of NE on the responsivity of the two SC neurons that we tested.

Development and plasticity of the serotoninergic projection to the hamster's superior colliculus.

Immunocytochemical techniques were employed to investigate the normal adult organization, development, and effects of both neonatal and adult eye removal upon the organization of the serotoninergic projection to the hamster's superior colliculus. Immunocytochemistry, both alone and in combination with retrograde tracing with true blue and fluorogold, was used to determine the organization of the serotoninergic projection to the superior colliculus in normal adult hamsters. Immunoreactive fibers were present in all laminae of the superior colliculus, but they were most dense in the lower part of the stratum griseum superficiale, the stratum opticum, the stratum griseum profundum, and the stratum album profundum. When retrograde tracing from the colliculus was combined with immunocytochemistry for serotonin, cells containing both labels were found in the lateral portions of the nucleus raphe dorsalis and also in periaqueductal gray, the median and pontine raphe nuclei, and in the region of the medial lemniscus. Such cells were visible both ipsilateral and contralateral to the injection site. Serotonin immunoreactive fibers were visible in the superior colliculus by embryonic day 14 (2 days prior to birth). On the day of birth, a small number of immunoreactive fibers were present just below the pial surface and others were generally oriented either parallel or orthogonal to the collicular laminae. At this age, there were also many immunoreactive fibers that crossed from one side of the brainstem to the other in the commissure of the superior colliculus. Some serotonin-positive axons appeared to terminate as growth cones in fetal and newborn hamsters. Over the next 2 weeks, the serotoninergic innervation of the SC increased in density and assumed the laminar distribution observed in adult animals. By this age, only a very few immunoreactive fibers were present in the commissure of the superior colliculus. Removal of one eye on either on the day of birth or in adulthood resulted in reorganization of the serotoninergic innervation of the partially deafferented colliculus. There was a marked increase in the density of serotonin-positive fibers in the upper stratum griseum superficiale on the side ipsilateral to the remaining eye. This change was apparent within 2 weeks after enucleation in either neonatal or adult animals. Quantitative analysis demonstrated that the magnitude of this effect was greater after adult enucleation than after neonatal eye removal. Both neonatal and adult enucleation also resulted in an increase in the density of the serotoninergic projection to the dorsal and ventral lateral geniculate nuclei ipsilateral to the remaining eye.