B. A. Flumerfelt

Biotinylated dextran : a versatile anterograde and retrograde neuronal tracer

A powerful and versatile axonal tracing method using biotinylated dextran, a novel analogue of biotin, is described. Pressure injection of varying volumes of 5% biotinylated dextran into various parts of the brain and spinal cord resulted in Golgi-like retrograde labeling and PHA-L-like anterograde labeling. The tracer filled the finest processes, revealing terminal axonal ramifications, distal dendrites and dendritic spines and excrescences. Extensive anterograde and retrograde labeling occurred in all pathways studied and in animals of all ages. Labeling appeared as early as 48 h and remained unchanged up to 14 days following injection. Biotinylated dextran can be detected easily with any avidin-conjugated marker for light and electron microscopic study. The resultant labeling can be combined readily with other morphological methods, such as tract tracing and/or immunocytochemical demonstration of endogenous substances. Biotinylated dextran is thus an efficient anterograde and retrograde tracer that can be combined with other neuroanatomical techniques to study details of synaptic interaction at all levels of dendritic organization.

Efferent connections of the A1 noradrenergic cell group: A DBH immunohistochemical and PHA-L anterograde tracing study

Immunohistochemical localization of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) was employed to reveal the anatomical organization of the A1 noradrenergic cell group in the caudal ventrolateral medulla oblongata of the rat. Subsequently, the supraspinal efferent axonal projections of A1 were investigated with a view to elucidating the anatomical substrates underlying its postulated function in central fluid and cardiovascular homeostasis. Within the caudal medulla, DBH-positive/PNMT-negative (noradrenergic) neurons were observed extending bilaterally through the ventrolateral medullary reticular formation from upper cervical spinal cord levels to the level of the area postrema. At the rostral pole of A1, its neurons intermingled with PNMT-immunoreactive perikarya of the more rostrally situated C1 adrenergic cell group. Discrete injections of the anterogradely transported plant lectin Phaseolus vulgaris leucoagglutinin (PHA-L) into A1 resulted in terminal labeling in a number of presumptive efferent target sites including the nucleus of the solitary tract, rostral ventrolateral medulla, dorsal parabrachial nucleus, Kolliker-Fuse nucleus, central grey, dorsomedial nucleus of the hypothalamus, perifornical region, zona incerta, lateral hypothalamus, paraventricular nucleus of the hypothalamus, supraoptic nucleus, bed nucleus of the stria terminalis, and organum vasculosum of the lamina terminalis. Tissue sections adjacent to those reacted for PHA-L were processed immunohistochemically for DBH to determine if anterogradely labeled terminals were localized in regions that demonstrated appropriate immunoreactivity. The majority of regions in which PHA-L terminal labeling was present also exhibited moderate to intense DBH activity. These experiments provide neuroanatomical evidence for direct efferent pathways from the A1 noradrenergic cell group to a number of supraspinal sites that have been reliably implicated in the neural circuitry underlying the central regulation of fluid and cardiovascular homeostasis. Furthermore, the results suggest a selective anatomical interrelation between A1 and sites in the basal forebrain and hypothalamus in which vasopressinergic neurons have been previously demonstrated. It is postulated that the noradrenergic A1 projections observed in this investigation represent the morphological substrate through which A1 exerts a significant influence on cardiovascular regulatory mechanisms.

An anterograde HRP-WGA study of aberrant corticorubral projections following neonatal lesions of the rat sensorimotor cortex

SummaryAnterograde transport of horseradish peroxidase — wheat germ agglutinin (HRP-WGA) was used to examine the effect of unilateral neonatal ablation of the sensorimotor cortex on the remaining corticofugal projections to the midbrain in the rat. In unlesioned animals, the sensorimotor cortical efferents to the midbrain were entirely ipsilateral, terminal labeling being evident in the red nucleus, the midbrain reticular formation, the periaqueductal gray, the intermediate gray layer of the superior colliculus, the nucleus parafascicularis prerubralis and the perilemniscal area. Corticorubral fibers were seen to reach the midbrain through the thalamus or the cerebral peduncle. In the red nucleus, terminal labeling was essentially restricted to the parvocellular region. In neonatally lesioned adults, aberrant corticofugal fibers crossed the midline to terminate in the contralateral red nucleus, the midbrain reticular formation, the periaqueductal gray, the nucleus parafascicularis prerubralis and the intermediate gray layer of the superior colliculus. The aberrant projections maintained the topographic specificity of the normal ipsilateral projections. This was most evident in the corticorubral projection, where the aberrant contralateral fibers terminated in the parvocellular area of the red nucleus.

The pallidostriatal projection in the rat: a recurrent inhibitory loop?

The pallidostriatal projection in the rat was investigated employing the PHA-L tracing technique. Following inotophoretic injections into the lateral aspect of the globus pallidus external segment, the ipsilateral striatum showed patches of dense anterograde labeling separated by areas containing sparse anterograde labeling and isolated retrogradely labeled neurons. The densely labeled patches did not correspond to any known compartments of the striatum. The retrogradely labeled neurons consistently showed similar distribution and morphological features reminiscent of striatal type II projection neurons. As all projection neurons of the striatum and all pallidal neurons are GABAergic, the complementary pattern of anterogradely and retrogradely labeled profiles from the globus pallidus suggest a possible mechanism whereby a horizontal inhibition may be exerted on groups of striatal neurons via the striato-pallido-striatal pathway.

Topographic specificity of aberrant cerebellorubral projections following neonatal hemicerebellectomy in the rat.

Anterograde transport of horseradish peroxidase-wheat germ agglutinin (HRP-WGA) was used to examine the topographic specificity of ascending cerebellar efferent projections in adult rats which were hemicerebellectomized at birth. The results were compared to similar cerebellar projections in unlesioned adults. HRP-WGA placement in the nucleus interpositus of control rats resulted in a dense projection of labeled fibers which decussated in the midbrain, caudal to the red nucleus. In the red nucleus, dense terminal labeling was confined to the magnocellular region, while retrogradely labeled rubrocerebellar neurons were present throughout both parvo- and magnocellular areas. Similar HRP-WGA placements in the nucleus lateralis gave rise to fewer labeled fibers which terminated in the parvocellular red nucleus. In addition to the cerebellorubral projection, other areas of terminal labeling included the mid-brain reticular formation, nucleus parafascicularis prerubralis, zona incerta, fields of Forel and ventral thalamus. In neonatally lesioned adults, aberrant cerebellorubral and cerebellothalamic projections were observed deflecting ipsilaterally at the decussation of the normal contralateral projection. Topographic specificity of the aberrant ipsilateral cerebellorubral projection mirrored that of the normal contralateral fibers. In addition, an ipsilateral projection from the cerebellum could be followed rostral to the red nucleus, to terminate in the ipsilateral ventral thalamus. Lesioned animals also demonstrated marked cell loss in the red nucleus contralateral to the hemicerebellectomy.

Glutamatergic regulation of haloperidol-induced c-fos expression in the rat striatum and nucleus accumbens.

Acute administration of haloperidol induces the expression of the immediate-early gene c-fos in the striatum and nucleus accumbens via dopamine D(2) receptor antagonism. Dopaminergic transmission in the striatum and nucleus accumbens is modulated by glutamate via N-methyl-D-aspartate (NMDA) receptors. Indeed, haloperidol-induced c-fos expression is dependent on NMDA receptor activation in the dorsolateral part of the striatum. However, the role that NMDA receptors play in haloperidol-induced c-fos expression in other functionally distinct areas of the striatum and nucleus accumbens has not yet been established. Therefore, in the present study the entire rostrocaudal extent of the rat striatum and nucleus accumbens was examined to determine the role that NMDA receptors play in haloperidol-induced c-fos expression. Pretreatment with MK-801, a non-competitive antagonist of NMDA receptors, significantly reduced the number of neurons showing c-fos immunoreactivity in the rostral aspect of the dorsolateral striatum and the entire rostrocaudal extent of the ventrolateral striatum following an acute injection of haloperidol. However, the same treatment did not modify the pattern of haloperidol-mediated c-fos expression in the medial or central parts of the striatum. Similarly, MK-801 pretreatment significantly suppressed the number of neurons expressing c-fos immunoreactivity following haloperidol injection in the entire rostrocaudal extent of the shell region of nucleus accumbens, but not in the core region. The results indicate that haloperidol-induced c-fos expression is dependent on NMDA receptors only in the rostral aspect of the dorsolateral striatum and the rostrocaudal extent of the ventrolateral striatum, the areas involved in motor function. The differential role that NMDA receptors play in modulating haloperidol-mediated dopamine D(2) receptor antagonism between motor and associative areas of the striatum may contribute to the development of extrapyramidal symptoms following chronic haloperidol treatment. Furthermore, the attenuation of the haloperidol-induced c-fos expression by MK-801 was restricted to the nucleus accumbens shell, an area often implicated in the therapeutic effect of haloperidol. Therefore, the NMDA-dopamine D(2) receptor interaction may also play a role in mediating the therapeutic effects of haloperidol.

A horseradish peroxidase study of the projections from the lateral reticular nucleus to the cerebellum in the rat

SummaryThe projection from the lateral reticular nucleus (LRN) to the cerebellar cortex was studied in the rat by utilizing the retrograde transport of horseradish peroxidase (HRP). In order to study the topographic features of this projection, small amounts of HRP were injected into various sites in the cerebellar cortex. The results demonstrated that the caudal lobules of the anterior lobe vermis tend to receive afferents from the medial LRN and the rostral lobules of the vermis receive afferents from more laterally situated cells. Lobules IV and V receive inputs primarily from the magnocellular division of the LRN of both the ventromedial and dorsolateral parts of the LRN, while lobules II and III receive inputs mainly from cells which lie in the border area between the parvocellular and magnocellular division of the ventromedial part. Following injections within various areas of the posterior lobe vermis, the results indicated that lobule VIII receives the most abundant projection from the LRN and that the cells of origin are present within the parvocellular and the adjacent part of the magnocellular division throughout the rostrocaudal extent of the LRN. Following injections within lobules VI and VII, few labelled cells were found and they tended to lie within the rostral two-thirds of the magnocellular division. Little or no projection from the LRN to lobule IX was evident. The hemispheres were found to receive a modest projection from the dorsal aspect of the LRN. The projection to lobulus simplex originates mainly from the caudal two-thirds of the magnocellular division, while the projection to the ansiform and paramedian lobules originates mainly from the dorsal aspect of the rostral two-thirds of the magnocellular division. Finally, there appears to be extensive overlapping of the orgins of all three projections to the cerebellar cortex studied, and this occurs within the central area of the magnocellular division throughout the rostrocaudal extent of the LRN.

Acetylcholinesterase histochemistry of the habenulo-interpeduncular pathway in the rat and the effects of electrolytic and kainic acid lesions

SummaryThe histochemical distribution of acetylcholinesterase (AChE) was studied in the habenulo-interpeduncular pathway of normal rats and after electrolytic and kainic acid lesions of the habenular nuclei. From these combined observations it appears that the AChE-rich projection to the interpeduncular nucleus derives from both the medial and the lateral habenular nuclei. The lateral nucleus of the habenula is the main source of AChE-rich fibres in the fasciculus retroflexus, and a number of stained fibres also derive from the stria medullaris. While total habenular lesions completely deprived the fasciculus retroflexus of AChE-stained fibres, a direct effect on the enzyme distribution in the interpeduncular nucleus was only apparent at its rostral pole. In the remainder of the nucleus the AChE distribution did not undergo obvious changes in comparison with the normal pattern, except for a moderate decrease in overall reaction intensity in cases with subtotal habenular lesions bilaterally.The above results are consistent with the observations derived from experiments involving kainic acid injection into the habenula. The neurotoxic effect of kainic acid was highly selective for specific types of neurons in the lateral habenula, while the neurons of the medial habenula were completely unaffected. The existence of an AChE-rich projection from the lateral habenula to the interpeduncular nucleus was supported by a corresponding decrease in enzyme activity in the lateral habenula and fasciculus retroflexus after kainic acid treatment.

Afferent organization of the lateral reticular nucleus in the rat: An anterograde tracing study

SummaryThe organization of the afferent projections to the lateral reticular nucleus of the rat was investigated following placement of horseradish peroxidase-conjugated wheatgerm agglutinin into the red nucleus, fastigial nucleus, various levels of the spinal cord or the sensorimotor area of the cerebral cortex. The pattern of distribution of anterogradely labelled profiles visualized with tetramethylbenzidine revealed that the caudal three-fourths of the lateral reticular nucleus received a large, topographically organized projection from the entire length of the contralateral spinal cord. The lateral part of the rostral half of the lateral reticular nucleus received a small projection from the contralateral red nucleus, the dorsal part of the middle third of the nucleus received a diffuse projection from the contralateral fastigial nucleus, and the extreme rostromedial part of the nucleus received a sparse projection from the contralateral cerebral cortex. The dorsal part of the middle third of the lateral reticular nucleus also received a small projection from the ipsilateral cervical spinal cord. The distribution of afferent fibres from different levels of the spinal cord, red nucleus, and fastigial nucleus overlapped substantially in the middle third of the lateral reticular nucleus, whereas the cerebral cortical receiving area was separate. These data suggest that the middle third of the lateral reticular nucleus integrates spinal and supraspinal impulses to the cerebellum, while the rostral part of the nucleus is involved in a separate cerebral cortico-cerebellar pathway.

Contralateral corticorubral fibers induced by neonatal lesions are not collaterals of the normal ipsilateral projection

Unilateral neonatal cortical ablation induces the development of a bilateral corticorubral projection from the remaining sensorimotor cortex. The retrograde fluorescent tracers Fast blue (FB) and Nuclear yellow (NY) were used to determine if the aberrant contralateral projection arises from axon collaterals of the normal uncrossed projection. Six to 8 weeks after unilateral cortical ablation in neonatal rats, the red nuclei were injected with FB on one side and NY on the other to study the source of the normal and aberrant afferents from the cerebral cortex. In control animals, many neurons in layer V of the sensorimotor cortex were retrogradely labeled with the tracer that had been injected into the ipsilateral red nucleus. In animals with unilateral ablations, many neurons throughout the remaining sensorimotor cortex were retrogradely labeled with FB or NY. No cortical neurons were doubly labeled. In addition to demonstrating the bilaterality of the corticorubral projection in animals which had received neonatal lesions, these results indicate that the aberrant contralateral corticorubral projection does not consist of axon collaterals of the normal ipsilateral fibers.