Nitric oxide-containing neurons in long-term grafts in a rat model of Parkinson's disease.

The role that nitric oxide may play in modulating graft function in long-term fetal ventral mesencephalic grafts in an animal model of Parkinson's disease was investigated. Mature grafts harvested from the entire fetal ventral mesencephalon possessed a large number of neuronal nitric oxide synthase (nNOS)/NADPH-diaphorase-containing neurons throughout the graft intermingled with dopaminergic neurons. The morphological and neurochemical characteristics of these NADPH-diaphorase neurons resembled those in centers adjacent to the substantia nigra of adult brain but not that of the striatum. Pretreatment with the nNOS blocker, 7-nitroindazole, resulted in contralateral rotations following methamphetamine challenge in long-term grafted animals that previously showed normalized rotational behavior. In contrast, mature grafts derived from fetal ventral mesencephalon without the midline areas possessed only a few nNOS-containing neurons within the grafts, and a similar methamphetamine challenge following 7-nitroindazole pretreatment in long-term grafted rats that previously showed normalized rotational behavior resulted in random movements. Our results indicate that nitric oxide-containing neurons inadvertently included during grafting may affect graft function, and excluding the midline areas of the ventral mesencephalon during tissue harvesting may minimize this effect.

Glutamatergic regulation of long-term grafts of fetal lateral ganglionic eminence in a rat model of Huntington's disease.

Transplanting fetal striatal tissue is currently considered to be an important alternative strategy in the treatment of Huntington's disease. Although grafted striatal tissue differentiates and shows certain structural and neurochemical features of the normal striatum and receives host afferents, it is not clear whether host-derived afferent inputs can modulate the activity of neurotransmitter receptors and their signaling in the graft. An intricate interaction between dopaminergic and glutamatergic systems is pivotal for striatal function. In the present study, the modulation of D(2) receptors in the graft by host-derived glutamatergic afferents via NMDA receptors was investigated using haloperidol-induced c-Fos expression. The results indicate that haloperidol induces c-Fos in a large number of neurons in the P-zones of the graft and this induction is significantly suppressed by pretreatment with the NMDA receptor antagonist, MK-801. Therefore, the NMDA receptor-mediated modulation of D(2) receptor function seen in the normal striatum is established in the striatostriatal grafts.

Muscarinic, adenosine A(2) and histamine H(3) receptor modulation of haloperidol-induced c-fos expression in the striatum and nucleus accumbens.

It is generally believed that haloperidol exerts its motor side effects and therapeutic effects mainly by antagonizing dopamine D(2) receptors in the striatum and the nucleus accumbens, respectively. Several neurotransmitters/modulators, including glutamate, acetylcholine, adenosine and histamine, affect dopaminergic activity in these centers. We have recently shown that N-methyl-D-aspartate receptor-mediated modulation of haloperidol-induced c-fos expression differs in functionally specific regions of the striatum and the nucleus accumbens. In the present study, the entire striatum and the nucleus accumbens were comprehensively examined for the pattern of modulation of haloperidol-induced c-fos expression by adenosine A(2), histamine H(3) and muscarinic receptor antagonists. Blockade of muscarinic and H(3) receptors resulted in a profound suppression of haloperidol-induced c-fos expression in the dorsolateral part of the striatum. In addition, the H(3) receptor antagonist suppressed the effects of haloperidol in the ventrolateral aspect of the striatum and the rostral parts of the medial striatum. Muscarinic receptor antagonists suppressed haloperidol-induced c-fos expression throughout the shell and in the mid-level of the core of the nucleus accumbens while A(2) and H(3) receptor antagonists did not.We found that the muscarinic and H(3) receptor antagonists suppress the induction of c-fos by haloperidol in the dorsolateral aspect of the striatum, an area implicated in the development of extrapyramidal motor symptoms following chronic haloperidol treatment. By contrast, haloperidol-induced c-fos expression in the nucleus accumbens, an area implicated in the therapeutic effects of haloperidol, was suppressed by the muscarinic receptor antagonist, but not by the H(3) receptor antagonist. Therefore we conclude that H(3) receptor modulation may provide a useful therapeutic target in future efforts to minimize neuroleptic-induced motor side effects.

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.

Changes in CArG-binding protein A expression levels following injection(s) of the D1-dopamine agonist SKF-82958 in the intact and 6-hydroxydopamine-lesioned rat.

We recently characterized the rat brain homolog of mouse muscle CArG-binding protein A initially identified in C2 myogenic cells and showed an inverse temporal correlation between increased expression levels of this messenger RNA, c-fos and zif268 messenger RNA levels following the addition of nerve growth factor to PC12 cells. In addition, we found an inverse correlation between c-Fos protein and CArG-binding protein A messenger RNA levels in the lateral caudate-putamen of rats treated acutely and chronically with the D2 receptor antagonist fluphenazine (phenothiozine typical psychotic). To determine whether D1 receptor stimulation is also capable of inducing CArG-binding protein A up-regulation, drug naive or dopamine-depleted (i.e. 6-hydroxydopamine-lesioned) D1 hypersensitized rats (i.e. rats given repeated daily injections of SKF-82958 for 14days) were acutely injected with the D1 agonist SKF-82958 and examined using a combination of in situ hybridization for CArG binding protein A and immunocytochemistry for c-Fos. Both acutely treated animals and dopamine-depleted hypersensitized animals showed increases in CArG-binding protein A. Moderate increases were found in the medial caudate-putamen and nucleus accumbens core and shell regions following acute treatment whereas large increases in CArG-binding protein A expression levels were found in the medial and lateral caudate-putamen and the shell and core of the nucleus accumbens following hypersensitization. No change in CArG-binding protein A expression level was found in the dopamine-depleted, drug naive animals relative to controls. Regions of the basal ganglia where increases in CArG-binding protein A were detected following each treatment correlated perfectly with c-Fos protein induction. The results demonstrate that CArG-binding protein A responds to SKF-82958 and that the changes in CArG-binding protein A match perfectly with the pattern of c-Fos induction induced by the D1 agonist.

Characterization of CArG-binding protein A initially identified by differential display.

While investigating differences in the pattern of gene expression in functionally distinct areas of the rat caudate-putamen employing differential display, we identified a gene that is highly enriched in tissue adjacent to the lateral ventricle. To characterize the gene, a complementary DNA containing the complete coding sequence was obtained and sequenced. In addition, radiolabelled DNA and riboprobes were generated to examine the expression levels and anatomical distribution of the identified gene in the brain. The sequencing data suggests that the identified gene is a member of the heterogeneous nuclear ribonucleoprotein family and likely represents the rat homolog of CArG-binding protein A initially isolated from mouse C2 myogenic cells. CArG-binding protein A is widely distributed and moderately expressed in the rat brain and present within both neurons and astrocytes. Since the CArG box motif forms the core of the serum response element and the serum response element is involved in immediate early gene regulation, the expression level of CArG-binding protein A was examined following treatment of PC12 cells with nerve growth factor and correlated with changes in c-fos and zif268 expression. The results show that CArG-binding protein A is up-regulated following nerve growth factor treatment and that the up-regulation of CArG-binding protein A can be correlated with the down-regulation of c-fos and zif268. The results of the current study leads us to suggest that CArG-binding protein A may be involved in brain development and the regulation of the serum response element.

A kappa opioid antagonist blocks sensitization in a rodent model of Parkinson's disease.

Treatment of Parkinson's disease with levodopa is associated with fluctuations in motor function and dyskinesias, which may in part depend upon the mode of levodopa treatment. In rats with unilateral 6-hydroxydopamine lesions, intermittent levodopa results in sensitization to apomorphine-induced rotation, associated with massive ipsilateral increases in nigral dynorphin. We assessed the effects of nigral infusion of the selective kappa opioid antagonist nor-binaltorphomine (nor-BNI) in this model. Nor-BNI reduced apomorphine-induced rotation in animals receiving intermittent levodopa to a level comparable with that seen in animals treated with continuous levodopa or with saline. These data suggest that behavioural sensitization arising from intermittent levodopa therapy in a rodent model of parkinsonism depends upon increased expression of dynorphin in the striatonigral pathway and provides further insight into the mechanisms underlying motor fluctuations which develop during the treatment of Parkinson's disease.

Effects of graft-derived dopaminergic innervation on the target neurons of patch and matrix compartments of the striatum.

Fetal dopaminergic neurons grafted into the dopamine-depleted striatum have previously been shown to normalize neurochemical and behavioural abnormalities. However, the extent of graft-induced recovery of striatal compartments, which differ in their ontogeny, neurochemical properties and function, is still not clear. The striosome and matrix compartments of the striatum provide a segregated projection to somatostatin-containing GABAergic neurons of the rostral part of the entopeduncular nucleus and somatostatin-negative GABAergic neurons of the caudal part of the entopeduncular nucleus, respectively. In the present study, preprosomatostatin and glutamate decarboxylase messenger RNA levels in the rostral and caudal parts of the entopeduncular nucleus were determined six and 18 months postgrafting in rats with complete recovery of rotational behaviour following apomorphine challenge, and in rats with unilateral 6-hydroxydopamine lesions or sham lesions and no grafts. Sections were processed for in situ hybridization using 35S-labelled cRNA probes for glutamate decarboxylase (67,000 mol. wt isoform; GAD67) and preprosomatostatin. Autoradiographs showed a marked increase in preprosomatostatin messenger RNA within the ipsilateral entopeduncular nucleus in 6-hydroxydopamine-lesioned rats, and a substantially lower increase six months postgrafting. At 18 months postgrafting, the preprosomatostatin messenger RNA levels were symmetrical within the entopeduncular nucleus. Unilateral depletion of striatal dopamine resulted in a moderate increase in GAD67 messenger RNA levels within the ipsilateral entopeduncular nucleus, along with a substantial decrease in GAD67 levels within the contralateral nucleus. By six months postgrafting, the GAD67 levels had decreased considerably within the ipsilateral entopeduncular nucleus, while the messenger RNA levels had returned to normal within the contralateral nucleus. Interestingly, at 18 months postgrafting, the GAD67 levels remained decreased within the ipsilateral entopeduncular nucleus and were significantly lower than the normal value. The results indicate that fetal nigral grafts placed within the dopamine-depleted striatum can restore the neurochemical alterations seen in striatal target areas such as the entopeduncular nucleus. This may form the neurochemical basis of graft-induced behavioural recovery, as the normalization of neurotransmitter messenger RNA levels in the entopeduncular nucleus reflects the restoration of overall activity in both direct and indirect striatal output pathways. The results also indicate that the graft-derived dopaminergic innervation restores the output of both striosome and matrix compartments of the striatum. The present results also showed a progressive recovery leading to over-compensation of neurotransmitter messenger RNA levels following grafting, perhaps indicating the importance of feedback regulation of grafted dopaminergic neurons by the host.

Somatostatin and the patch/matrix compartments of the rat caudate-putamen.

In the caudate-putamen of the rat, two subpopulations of medium aspiny neurons exist that contain somatostatin. The first subpopulation contains somatostatin 14, somatostatin 28, and somatostatin 28(1-12). The other subpopulation contains only somatostatin 28. To examine the relationship between somatostatin-containing neurons and the patch/matrix compartments, a series of double-labelling experiments using antisera directed against different somatostatin peptides and calbindin were used. Sections stained in this manner were examined with the aid of a confocal microscope. The results of these experiments indicate that somatostatin 28(1-12)-containing neurons may play a role in matrix integration, with some input directed from the patch compartment. In addition, somatostatin 28-containing neurons are more numerous in the patch compartment than somatostatin 28(1-12)-containing neurons, suggesting a possible role for these neurons in patch integration.

Colocalization of somatostatin, neuropeptide Y, and NADPH-diaphorase in the caudate-putamen of the rat.

Somatostatin, neuropeptide Y, and nicotinamide adenine dinucleotide phosphate-diaphorase are colocalized within a small population of medium aspiny neurons in the caudate-putamen of the rat. The extent of colocalization, however, appears to be in dispute. In order to examine the question of colocalization between these three neuroactive substances, a series of double-labelling experiments was performed. This was accomplished by combining immunocytochemistry for somatostatin or neuropeptide Y or enzyme histochemistry for nicotinamide adenine dinucleotide phosphate-diaphorase with in situ hybridization for somatostatin and/or neuropeptide Y mRNA. The results of such analysis indicate that nicotinamide adenine dinucleotide phosphate-diaphorase and somatostatin mRNA are 100% colocalized throughout the caudate-putamen, except for the area bordering the globus pallidus. All neurons that contain neuropeptide Y contain somatostatin message. Only 84% of the neurons that contain somatostatin mRNA, however, also contain neuropeptide Y. Neurons that contain somatostatin 28 but not neuropeptide Y are found throughout the caudate-putamen. These results indicate that the somatostatin neuron population in the rat caudate-putamen is not homogeneous. Instead, the medium aspiny neuron population is actually composed of several subpopulations based on the content of neuroactive substances.

Parvalbumin-containing GABAergic neurons in the basal ganglia output system of the rat.

The output of the basal ganglia is directed through the entopeduncular nucleus (EPN) and the substantia nigra pars reticulata (SNR) and pars lateralis (SNL), which provide a gamma-aminobutyric acidergic (GABAergic) projection to various nuclei of the thalamus and brainstem. Although many neurons within the SNR and EPN have been described as modality specific, the morphological and neurochemical similarities preclude their precise identification. In the present study, the immunocytochemical localization of parvalbumin, a calcium-binding protein, is used in combination with axonal tracing to verify neuronal heterogeneity within the SNR, SNL, and EPN. The results reveal that the majority of neurons in all three centers contain parvalbumin. The parvalbumin-containing neurons are distributed in the caudal two-thirds of the EPN, the rostral part of the SNL, and the lateral two-thirds of the entire rostrocaudal extent of the SNR, the areas involved in sensorimotor function of the basal ganglia. Moreover, the nigrothalamic, nigrocollicular, and EPN-thalamic neurons possess parvalbumin immunoreactivity, whereas the EPN-habenular neurons are devoid of parvalbumin immunoreactivity. The results indicate a neurochemical heterogeneity within the GABAergic output neurons of the basal ganglia and suggest that the parvalbumin-containing neurons of the SNR, SNL, and EPN are the tonically active output neurons that form a major link in the disinhibitory neuronal circuit of the basal ganglia, especially that concerned with sensorimotor function.

Colocalization of prosomatostatin-derived peptides in the caudate-putamen of the rat.

In the striatum of rat, somatostatin 14, somatostatin 28, and somatostatin 28(1-12) have previously been localized within a small population of medium aspiny local circuit neurons. Because all three peptide fragments are generated through the cleavage of prosomatostatin by different converting enzymes, the possibility for differential expression of these peptides exists. In order to investigate this possibility, frozen sections were collected from the brains of adult female Wistar rats fixed with 4% paraformaldehyde and double labelled using immunocytochemistry and in situ hybridization. Sections were first processed for somatostatin 14, somatostatin 28, or somatostatin 28(1-12) by using the avidin-biotin complex immunocytochemical technique followed by in situ hybridization using 35S-labelled antisense riboprobes to somatostatin mRNA. The results of such analysis revealed that somatostatin 28 and somatostatin mRNA are 100% colocalized. Somatostatin 14 and somatostatin 28(1-12), in contrast, are only present within 66% of the neurons that express somatostatin mRNA. Examination of the anatomical distribution of neurons that express both somatostatin mRNA and somatostatin 14 or somatostatin 28(1-12) protein reveals that these neurons are present throughout the caudate-putamen of rat but are more prevalent in the ventromedial regions. Neurons that express somatostatin mRNA but not somatostatin 14 or somatostatin 28(1-12) are also present throughout the caudate-putamen but are most numerous within a dorsolateral strip just beneath the corpus callosum. These results suggest that the somatostatin neuron population within the rat caudate-putamen is actually composed of two smaller subpopulations based on neuropeptide content. The first subpopulation contains somatostatin 28 and constitutes one-third of the total somatostatin population, whereas the other contains somatostatin 28, somatostatin 14, and somatostatin 28(1-12) and represents the remaining two-thirds of the cells that express somatostatin mRNA.

Neurochemical compartmentalization of the globus pallidus in the rat: an immunocytochemical study of calcium-binding proteins.

The globus pallidus external segment forms a major target center of the mammalian striatum which is characterized by neurochemically distinct compartments. The present study was undertaken to determine if a corresponding compartmentalization exists within the globus pallidus external segment in the rat. Immunocytochemical examination of the calcium-binding proteins parvalbumin and calbindin D28kDa, which are present in neurons of the striatal matrix compartment, was employed. The results indicate three neurochemically distinct compartments within the globus pallidus external segment: 1) an area in the medial aspect of the entire length of the globus pallidus that contains dense immunoreactivity for calbindin D28kDa; 2) a narrow rim at the striatopallidal junction in the rostral two-thirds of the globus palidus that contains calbindin D28kDa immunoreactivity designated as the "border zone" of the globus pallidus; and 3) an area between these two zones showing very poor immunoreactivity for calbindin D28kDa but containing parvalbumin immunoreactive neurons. The calbindin D28kDa immunoreactive border zone corresponds to the area of the globus pallidus where striatal inputs converge extensively, whereas the rest of the nucleus is involved in segregated, topographically organized pathways. Parvalbumin-containing neurons are involved in the propagation of striatal output related to striosomal and sensorimotor aspects of basal ganglia function. The present results also indicate that calbindin D28kDa immunoreactivity is completely absent from striosomal neurons and is therefore a useful marker for striatal compartments.

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.

GABAergic synaptic interactions in the substantia nigra.

The substantia nigra receives a strong GABAergic input from the ipsilateral striatum and globus pallidus. Nigral GABAergic synaptic interactions have been described in the pars compacta (SNC) and pars reticulata (SNR) but not in the pars lateralis (SNL). The SNR and particularly the SNL are the nodal points of the GABAergic nigrotectal pathway. The present study analyzes the synaptic connections of GABAergic and dopaminergic neurons in each of the divisions of the substantia nigra by employing a double-labeling immunocytochemical technique at the light and electron microscope levels. Glutamic acid decarboxylase (GAD)-containing terminals make symmetrical synaptic contacts with dopaminergic neurons in the SNC and SNR. Neurons that contain GAD also receive a GABAergic input in the SNR and SNL. The proportion of GAD-GAD contacts appears to be highest in the SNL where virtually all GAD-positive terminals are found to be in synaptic contact with or apposed to GAD positive profiles. This study demonstrates a strong GABAergic input onto nigral dopaminergic neurons and GABAergic neurons in the SNR and SNL. This GABAergic influence which is ostensibly striatal or pallidal in origin is particularly prominent in relation to the SNL-mediated nigro-collicular pathway.

Compartmental origin of the striato-entopeduncular projection in the rat.

The mammalian neostriatum is divisible into neurochemically and cytoarchitectonically distinct striosome and matrix compartments. This compartmentalization is respected by many afferent and efferent projections of the striatum. The distribution of distinct types of neuroactive substances and receptors and the unique connections of the striosome and matrix suggest a functional segregation between these compartments. The present study examines the organization of efferent projections from each of the striatal compartments to the entopeduncular nucleus (EPN), a major output center of the basal ganglia. The fluorescent retrograde tracer fluorogold, or rhodamine-conjugated dextran, was injected into the lateral habenula or the ventrolateral nucleus of the thalamus of adult Wistar rats to identify the topographical organization of EPN-habenular and EPN-thalamic neurons. Fluorogold was then placed into the rostral or caudal parts of the EPN, identified from the previous experiment as areas containing predominantly EPN-habenular or EPN-thalamic neurons, respectively. Sections containing retrogradely labeled neurons in the neostriatum were simultaneously immunolabeled for calbindin-D28kDa, a calcium-binding protein found exclusively in the projection neurons of the matrix. The results indicate that the striatal projection to the EPN-habenular and EPN-thalamic parts of the EPN originates from striosome and matrix neurons, respectively. The duality of striatal outflow involving the EPN suggests a mechanism whereby the striosome is integrated into subcortical pathways that modulate the activity of the basal ganglia via the ascending serotoninergic projection from the dorsal raphe nucleus, whereas the matrix is involved in a loop that includes the thalamus and the cerebral cortex.

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.

Normalization of striatal proenkephalin and preprotachykinin mRNA expression by fetal substantia nigra grafts.

The expression of striatal proenkephalin and preprotachykinin mRNA was studied in Wistar rats following unilateral injections of 6-hydroxydopamine into the nigrostriatal pathway and after grafting dopaminergic fetal cell suspensions into the dopamine-depleted striatum. Striatal dopaminergic deafferentation resulted in an amphetamine-induced rotational asymmetry, an increase in ipsilateral striatal proenkephalin mRNA, and a decrease in preprotachykinin mRNA expression. Twelve months following grafting, proenkephalin and preprotachykinin mRNA returned to near-normal levels in contrast to control nongrafted lesioned animals. In addition, reversal of the rotational asymmetry was routinely observed. This study has demonstrated long-term graft-induced functional recovery coincident with normalization of striatal proenkephalin and preprotachykinin mRNA expression.

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

The 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.

Substance P immunoreactivity in the rat interpeduncular nucleus: synaptic interactions between substance P-positive profiles and choline acetyltransferase- or glutamate decarboxylase-immunoreactive structures.

The subnuclear and synaptic distribution of substance P immunoreactivity was examined in the rat interpeduncular nucleus at the light and electron microscope level. The nucleus possessed a prominent substance P-immunoreactive axonal plexus in the lateral and dorsomedial subnuclei, and in the dorsal cap of the rostral subnucleus. The density of substance P-immunoreactive axons in the remaining subnuclear divisions was sparse to moderate. Terminals of immunoreactive axons contained spherical vesicles and formed asymmetric contacts on dendritic processes exclusively. Immunoreactive neurons, restricted to the rostral subnucleus, possessed long, sparsely branched dendrites. Unlabelled terminals containing either spherical or pleomorphic vesicles contacted substance P-immunoreactive dendritic profiles. Axodendritic and axosomatic synapses containing substance P immunoreactivity pre- and postsynaptically were not observed. Ultrastructural evidence for synaptic relationships between substance P-containing profiles and those containing either choline acetyltransferase or glutamate decarboxylase was obtained by means of double antigen immunohistochemistry. Terminals of fasciculus retroflexus axons stained for choline acetyltransferase immunoreactivity formed asymmetric synaptic contacts with substance P-immunoreactive dendritic profiles. Few substance P-positive dendrites in the rostral subnucleus received terminals possessing glutamate decarboxylase activity. Unlabelled terminals containing either spherical or pleomorphic vesicles contacted substance P- and glutamate decarboxylase-immunoreactive dendritic profiles simultaneously. Terminals possessing either substance P or glutamate decarboxylase immunoreactivity formed synaptic contacts with dendritic processes of neurons in the lateral subnucleus. Many of the neurons within this subnuclear division contained glutamate decarboxylase. This study provides direct evidence of synaptic relationships between choline acetyltransferase-immunoreactive axons and substance P-immunoreactive dendritic profiles, and between substance P-positive axons and glutamate decarboxylase-immunoreactive dendrites. These findings reveal that two types of transmitter-specific axons of the fasciculus retroflexus innervate neuronal populations of the interpeduncular nucleus stained immunohistochemically for either substance P or glutamate decarboxylase.