Nicolas L. Chiaia

Postnatal blockade of cortical activity by tetrodotoxin does not disrupt the formation of vibrissa-related patterns in the rat's somatosensory cortex.

Neuronal activity has been shown to influence pattern formation in the visual system. In the present study, we determined whether or not this was also true in the somatosensory system by silencing the primary somatosensory cortex of rats with tetrodotoxin (TTX) for the first 7-11 days of life. Application of TTX during this period did not prevent the formation of the normal vibrissa-related pattern in S-I as visualized by either staining cortical sections for cytochrome oxidase, demonstration of the pattern with an antibody directed against serotonin, or labelling of thalamocortical axons with the carbocyanine dye, Di-I. These results indicate that neither peripherally evoked nor spontaneous activity are required for qualitatively normal pattern formation in the rats primary somatosensory cortex.

Intersubnuclear Connections within the Rat Trigeminal Brainstem Complex

Prior intracellular recording and labeling experiments have documented local-circuit and projection neurons in the spinal trigeminal (V) nucleus with axons that arborize in more rostral and caudal spinal trigeminal subnuclei and nucleus principalis. Anterograde tracing studies were therefore carried out to assess the origin, extent, distribution, and morphology of such intersubnuclear axons in the rat trigeminal brainstem nuclear complex (TBNC). Phaseolus vulgaris leucoagglutinin (PHA-L) was used as the anterograde marker because of its high sensitivity and the morphological detail provided. Injections restricted to TBNC subnucleus caudalis resulted in dense terminal labeling in each of the more rostral ipsilateral subnuclei. Subnucleus interpolaris projected ipsilaterally and heavily to magnocellular portions of subnucleus caudalis, as well as subnucleus oralis and nucleus principalis. Nucleus principalis, on the other hand, had only a sparse projection to each of the caudal ipsilateral subnuclei. Intersubnuclear axons most frequently traveled in the deep bundles within the TBNC, the V spinal tract, and the reticular formation. They gave rise to a number of circumscribed, highly branched arbors with many boutons of the terminal and en passant types. Retrograde single- or multiple-labeling experiments assessed the cells giving rise to TBNC intersubnuclear collaterals. Horseradish peroxidase (HRP) and/or fluorescent tracer injections into the thalamus, colliculus, cerebellum, nucleus principalis, and/or subnucleus caudalis revealed large numbers of neurons in subnuclei caudalis, interpolaris, and oralis projecting to the region of nucleus principalis. Cells projecting to more caudal spinal trigeminal regions were most numerous in subnuclei interpolaris and oralis. Some cells in lamina V of subnucleus caudalis and in subnuclei interpolaris and oralis projected to thalamus and/or colliculus, as well as other TBNC subnuclei. Such collateral projections were rare in nucleus principalis and more superficial laminae of subnucleus caudalis. TBNC cells labeled by cerebellar injections were not double-labeled by tracer injections into the thalamus, colliculus, or TBNC. These findings lend generality to currently available data obtained with intracellular recording and HRP labeling methods, and suggest that most intersubnuclear axons originate in TBNC local-circuit neurons, though some originate in cells that project to midbrain and/or diencephalon.

Trigeminal projections to contralateral dorsal horn: central extent, peripheral origins, and plasticity.

Prior studies have documented a trigeminal (V) mandibular primary afferent projection to the dorsomedial portion of the contralateral medullary and cervical dorsal horns in cat, hamster, and rat. We now report the existence of a much more substantial V ophthalmic primary afferent projection to the ventrolateral portion of contralateral medullary and cervical dorsal horns in rat. Horseradish peroxidase (HRP) injections into the V ganglion or V brainstem complex anterogradely labeled a fascicle of primary afferent axons that exited the caudal ventrolateral V spinal tract to form a rostrocaudally continuous, transversely oriented, V primary afferent decussation. These fibers terminated most heavily in laminae III-V of the ventrolateral dorsal horn in contralateral caudal medulla and the first and second cervical segments. Retrograde tracing with diamidino yellow (DY) or fluorogold and anterograde tracing with Phaseolus vulgaris leucoagglutinin also demonstrated a substantial commissural projection of central origin in medullary dorsal horn laminae I-VII. The latter projection had a more diffuse trajectory and termination pattern than that of the V primary afferent decussation. Unilateral HRP injections into medullary and cervical dorsal horns also retrogradely labeled V primary afferent collaterals contralateral to the injection site in corresponding regions of dorsal horn, and also in ventromedial interpolaris, oralis, and principalis, rostral to their decussation. Axons (1.5 +/- 0.8 microns mean diameter; 0.4-3.9 microns range) therefore terminated both ipsi- and contralateral to their cells of origin. These HRP injections also labeled an average of 40.4 +/- 13.0 V ganglion cells (mean +/- SD, corrected for split somata) in dorsomedial, ophthalmic regions of the contralateral ganglion. Their mean diameter was slightly larger than that of cells labeled ipsilaterally (29.9 vs. 26.3 microns). Double-labeling studies assessed possible ophthalmic receptor surfaces innervated by centrally crossing primary afferents. DY was injected into right medullary and cervical dorsal horns, and HRP was applied to either the left cornea, the ethmoid nerve, or the dura overlying cerebral cortex. Though DY labeled from 75 to 125 left ganglion cells per animal, no cells were double-labeled. All of these findings suggest that nociceptive-specific ganglion cells are not a source of the crossed ophthalmic primary afferent projection. Unilateral transection of the infraorbital nerve on the day of birth did not alter the crossed primary afferent projection to the partially deafferented side of the brainstem. This is further evidence of an absence of central sprouting in spared V primary afferents following neonatal V deafferentation.

Hemorrhage induces Fos immunoreactivity in rat medullary catecholaminergic neurons.

In urethane anesthetized rats one hour after lowering the systolic blood pressure to 70-75 mmHg by withdrawing 3-4 ml of blood, Fos immunoreactivity (Fos-IR), confined to the cell nucleus, was detected bilaterally in numerous cells of the nucleus of the solitary tract (NTS) and ventrolateral medulla (VLM). A few Fos-IR neurons were observed in the lateral reticular nucleus, dorsal medullary reticular nucleus, spinal trigeminal nucleus, medial inferior olive, interfasciculus hypoglossi and paramedian rostral medulla. In sham-operated animals, a much smaller number of Fos-IR neurons were scattered in the NTS, VLM and other nuclei mentioned above. Double labeling with antisera to tyrosine-hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT) showed that 60% of TH-positive neurons in the NTS contained Fos-IR, and 70-80% of TH-positive neurons in the caudal VLM and 50-60% of PMNT-positive neurons in the rostral VLM expressed Fos-IR. Only a few TH- or PNMT-positive neurons in the C2, C3 (paramedian rostral medulla) areas and within the medial longitudinal fasciculus were Fos-IR. About 40% of PNMT/Fos-IR neurons in the rostral VLM contained the retrograde tracer fluorogold, which was injected (< 1 microliter) into the white matter dorsolateral to the intermediolateral cell column of T2-T3 segments 2 to 3 days prior to hemorrhagic experiments. Very few TH-positive neurons in the caudal VLM contained fluorogold. Finally, clusters of Fos-IR neurons, which also labeled with antisera to choline acetyltransferase, were detected in the intermediolateral cell column of the spinal cord. The results indicate that during hemorrhage aminergic neurons in the caudal and rostral VLM and in the NTS are activated insofar as c-fos expression is concerned. As a corollary, the monoaminergic neurons in the medulla constitute an essential component in the ascending as well as descending reflex pathway involved in the adjustment of cardiovascular dynamics during hemorrhage.

Thalamocortical afferents in rat transiently express high-affinity serotonin uptake sites.

Autoradiographic techniques using [3H]citalopram were employed in 8-day-old (P-8) and adult rats to delineate the distribution of high-affinity serotonin (5-HT) uptake sites in the cerebral cortex. In the postnatal rats, [3H]citalopram binding sites were densely distributed in the lower portion of layer III, lamina IV, and upper layer V in the primary visual, somatosensory, and auditory cortices. In the primary somatosensory cortex, these binding sites were arrayed in a manner exactly matching the representation of the body surface as demonstrated by other methods such as staining for cytochrome oxidase (CO) or acetylcholinesterase (AChE). In adult rats, there was no differential distribution of [3H]citalopram binding sites in the cerebral cortex. Neonatal administration of the 5-HT neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), resulted in a nearly complete destruction of the 5-HT innervation of the cortex on P-8, but the patterned distribution of [3H]citalopram binding sites remained visible. In contrast, thalamic lesions carried out on P-4 caused a complete loss of the patterned distribution of [3H]citalopram binding sites in rats killed on either P-5 or P-8. These results are consistent with the conclusion that thalamocortical afferents in postnatal rats transiently express high-affinity uptake sites for 5-HT and thus may accumulate this amine.

Effects of postnatal blockade of cortical activity with tetrodotoxin upon lesion-induced reorganization of vibrissae-related patterns in the somatosensory cortex of rat

Previous studies have shown that postnatal blockade of thalamocortical activity with either tetrodotoxin (TTX) or the NMDA receptor antagonist DL-2-amino-5-phosphonovalerate (APV) does not prevent the formation of vibrissae-related patterns. In the present study, blockade of cortical activity with TTX was combined with ablation of a row of vibrissae follicles or transection of the infraorbital nerve (ION, the trigeminal nerve branch that supplies the vibrissae follicles) to determine whether the cortical reorganization that follows these lesions in otherwise untreated animals was dependent upon neuronal activity that could be blocked with TTX. The results demonstrated that cortical TTX implants had no quantitative or qualitative effects upon the cortical reorganization that followed either vibrissae follicle cauterization or ION transection.

Effects of Postnatal Blockade of Cortical Activity with Tetrodotoxin upon the Development and Plasticity of Vibrissa-Related Patterns in the Somatosensory Cortex of Hamsters

Several previous studies have shown that postnatal blockade of thalamocortical activity with either tetrodotoxin (TTX) or the N-methyl-D-aspartate (NMDA) receptor antagonist D,L-2-amino-5-phosphonovalerate (APV) does not prevent the formation of vibrissa-related patterns in the primary somatosensory cortex of rats. One limitation of these studies is that this pattern forms very shortly after birth in rats, and there may be only a very limited time over which it may be influenced by activity blockade. In the present study, the effect of activity blockade was evaluated in a more altricial rodent, the hamster. The present study showed that a pattern of thalamocortical afferents corresponding to the vibrissae is not observed until the fourth postnatal day in hamsters. Nevertheless, application of TTX-impregnated implants to the cortices of newborn hamsters had no qualitative or quantitative effect upon vibrissa-related patterns in the primary somatosensory cortices of these animals. Moreover, TTX implants did not prevent the changes in patterns that followed cauterization of a row of vibrissa follicles.

A substance P projection from the superior colliculus to the parabigeminal nucleus in the rat and hamster

Immunocytochemical staining with antisera directed against substance P (SP) demonstrated the existence of numerous immunoreactive neurons throughout the mediolateral and rostrocaudal extents of the stratum griseum superficiale (SGS) of the superior colliculus (SC) of both rat and hamster. In both of these species, very dense SP-like immunoreactivity (SPLI) was also visible in the parabigeminal nucleus. Combination of retrograde tracing with True blue or Fluorogold and immunocytochemistry demonstrated that SP-positive SC neurons projected to the parabigeminal nucleus in both hamster and rat. Retrogradely labelled and double-labelled cells were most numerous in the rostromedial portion of the SC and rare in the caudal portion of the colliculus. Destruction of the superficial layers of the SC resulted in a virtually complete loss of SPLI in the ipsilateral parabigeminal nucleus in both species. SPLI was also visible in two other targets of the superficial SC laminae: the intergeniculate leaflet and the ventral lateral geniculate nucleus. Ablation of the dorsal SC laminae did not reduce SPLI in either of those nuclei. Our results thus indicate that at least some tectoparabigeminal neurons in hamster and rat contain SPLI and further that the SC appears to be the sole source of SP-positive input to this nucleus.

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.