Hippocampal and midline thalamic fibers and terminals in relation to the choline acetyltransferase-immunoreactive neurons in nucleus accumbens of the rat: a light and electron microscopic study

The synaptic interactions between terminals of allocorticostriatal and thalamostriatal fibers and the cholinergic neurons in the nucleus accumbens were investigated using degeneration and dual labelling immunocytochemistry in Wistar rats. The presumptive cholinergic neurons were labelled with antibodies directed against choline acetyltransferase and the afferent fibers were labelled anterogradely with Phaseolus vulgaris-leucoagglutinin. Fibers from the subiculum of the hippocampal formation and from the midline and intralaminar thalamus project densely into the medial nucleus accumbens where they overlap a relatively dense population of choline acetyltransferase-immunoreactive neurons. Varicosities containing Phaseolus vulgaris-leucoagglutinin juxtapose the immunoreactive neurons. To study the possibility that the cholinergic neurons could be the synaptic targets of these incoming fibers, the subiculum, the fornix, and the midline/intralaminar thalamus were lesioned in separate animals and brain sections were immunoprocessed for choline acetyltransferase and studied with the electron microscope. In addition, dual-labelling electron microscopic immunocytochemistry was employed. In total, 164 synaptic terminals from the subiculum/hippocampus and 130 from the midline/intralaminar thalamus were examined; all formed asymmetrical synaptic specializations. No hippocampal endings were seen to contact the somata or primary dendrites of the choline acetyltransferase-immunoreactive neurons; however, three were found in synaptic contact with distal, immunolabelled dendritic shafts. Most hippocampal terminals established contacts with unlabelled spines. Fifteen percent of the thalamic endings were found to synapse on the somata and the primary and distal dendrites of the choline acetyltransferase-immunoreactive neurons. The remaining thalamic terminals established synaptic junctions with small unlabelled dendrites or spines. These findings have important implications not only for our understanding of the synaptic organization of the hippocampal and thalamic projections to the nucleus accumbens, but also for the contribution of the cholinergic neurons to the circuitry of this nucleus.     

Cholinergic innervation of the human thalamus: dual origin and differential nuclear distribution.

The cholinergic innervation of the human thalamus was studied with antibodies against the enzyme choline acetyltransferase (ChAT) and nerve growth factor receptor (NGFr). Acetylcholinesterase histochemistry was used to delineate nuclear boundaries. All thalamic nuclei displayed ChAT-positive axons and varicosities. Only the medial habenula contained ChAT-positive perikarya. Some intralaminar nuclei (central medial, central lateral, and paracentral), the reticular nucleus, midline nuclei (paraventricular and reuniens), some nuclei associated with the limbic system (anterodorsal nucleus and medially situated patches in the mediodorsal nucleus) and the lateral geniculate nucleus displayed the highest density of ChAT-positive axonal varicosities. The remaining sensory relay nuclei and the nuclei interconnected with the motor and association cortex displayed a lower level of innervation. Immunoreactivity for NGFr was observed in cholinergic neurons of the basal forebrain but not in cholinergic neurons of the upper brainstem. The contribution of basal forebrain afferents to the cholinergic innervation of the human thalamus was therefore studied with the aid of NGFr-immunoreactive axonal staining. The anterior intralaminar nuclei, the reticular nucleus, and medially situated patches in the mediodorsal nucleus displayed a substantial number of NGFr-positive varicose axons, presumably originating in the basal forebrain. Rare NGFr-positive axonal profiles were also seen in many of the other thalamic nuclei. These observations suggest that thalamic nuclei affiliated with limbic structures and with the ascending reticular activating system are likely to be under particularly intense cholinergic influence. While the vast majority of thalamic cholinergic input seems to come from the upper brainstem, the intralaminar and reticular nuclei, and especially medially situated patches within the mediodorsal nucleus also appear to receive substantial cholinergic innervation from the basal forebrain.     

Compartmental ordering of cholinergic innervation in the mediodorsal nucleus of the thalamus in human brain

The cholinergic innervation of the mediodorsal (MD) nucleus of the thalamus was visualized immunohistochemically in human brain postmortem, using an antibody against human choline acetyltransferase (ChAT). The ChAT staining of the MD nucleus was more intense than in the surrounding thalamic nuclei but weaker than that of the striatum. No ChAT-positive cell bodies were detected. The ChAT-positive neuropil was unevenly distributed, with patches of dense immunoreactivity contrasting with a weaker surrounding matrix. In adjoining sections stained for ChAT immunoreactivity and for acetylcholinesterase (AChE) activity, the zones enriched in ChAT-immunostained neuropil corresponded to AChE-rich regions. The three-dimensional reconstruction of the richest zone in AChE/ChAT activity evidenced a cylindrical organization throughout the rostrocaudal axis of the MD nucleus. Counts of ChAT-positive varicosities confirmed an inhomogeneous distribution; the density of varicosities was 30% higher in ChAT-rich regions than in surrounding matrix. These findings suggest that the activity of intrinsic neurons within the nucleus may be differentially regulated by cholinergic systems.     

Cholinergic innervation of the monkey amygdala: an immunohistochemical analysis with antisera to choline acetyltransferase

The organization of the cholinergic innervation of the macaque monkey amygdaloid complex was investigated by means of immunohistochemical techniques and either a polyclonal antiserum or a monoclonal antibody directed against the specific synthetic enzyme choline acetyltransferase (ChAT). Adjacent series of sections were processed histochemically for the demonstration of the degradative enzyme acetylcholinesterase (AChE) or for cell bodies with thionin. The density of ChAT immunoreactivity differed substantially among the various nuclei and cortical regions of the amygdala. In general, the distribution of ChAT immunoreactivity paralleled the pattern of AChE staining. One notable exception was the presence of AChE containing cell bodies in addition to AChE positive fibers within nearly all of the nuclear and cortical regions. In contrast, ChAT immunoreactivity was associated only with fibers and terminals. The highest density of ChAT immunoreactive fibers and terminals was consistently observed in the magnocellular subdivision of the basal nucleus. Staining was substantially less dense in the more ventrally situated parvicellular subdivision. Medially, in the adjacent accessory basal nucleus, immunoreactive fibers and terminals were densest in the magnocellular and superficial subdivisions and least prominent in the parvicellular subdivision. Of the deep nuclei, the lateral nucleus generally obtained the least ChAT immunoreactive terminals and processes. Only its more densely cellular ventrolateral portion contained appreciable fiber and terminal staining. One of the more distinctive patterns of ChAT immunoreactivity was seen in the nucleus of the lateral olfactory tract. Here, ChAT positive fibers formed pericellular basket plexuses around unstained cell bodies. This unique pattern of staining was used to delineate the boundaries of the nucleus and indicated that it is present for much of the rostrocaudal extent of the amygdala. Another region of conspicuous staining on the medial surface of the amygdala was the sulcal portion of the periamygdaloid cortex. This region, associated with the sulcus semiannularis and bordering the entorhinal cortex, consistently contained dense immunoreactivity. The central nucleus also presented a somewhat idiosyncratic pattern of ChAT staining. The lateral subdivision had a diffuse distribution of immunoreactivity in which focal patches of more densely stained terminals and occasional fine fibers were embedded. In contrast, the medial subdivision contained a larger number of thicker, stained fibers without diffuse background labeling.(ABSTRACT TRUNCATED AT 400 WORDS)     

Serotoninergic innervation of the thalamus in the primate: an immunohistochemical study.

Little is known of the serotoninergic innervation of the thalamus in primates; therefore, we undertook a detailed study of the distribution of 5-hydroxytryptamine (5-HT)-immunoreactive neuronal profiles in the thalamus of the squirrel monkey (Saimiri sciureus) with a specific antibody directly raised against 5-HT. All thalamic nuclei in the squirrel monkey displayed 5-HT-immunoreactive fibers, but none contained immunopositive cell bodies. The 5-HT innervation of the thalamus derived from extrinsic fibers arising mostly from the midbrain raphe nuclei and forming the transtegmental system. Most of the fibers destined to the thalamus collected into a major bundle that swept dorsoventrally within the midbrain tegmentum and coursed beneath the thalamus along its entire caudorostral extent. Several fiber fascicles broke off from this main bundle at different levels and ascended dorsally to innervate the various thalamic nuclei. Overall, the 5-HT innervation of the thalamus in the squirrel monkey was more massive than would have been expected from earlier studies in nonprimate species. Marked differences in the regional density of innervation were noted both between the various nuclei and within single nuclei. The most densely innervated nuclei were those delineating the principal subdivisions of the thalamic mass, that is, the midline, rostral intralaminar, limitans, and reticular nuclei, where very dense fields of isolated axonal varicosities occurred. In contrast to the rostral intralaminar nuclei, which were rather uniformly innervated, the centre médian/parafascicular complex contained immunoreactive fibers and isolated varicosities distributed according to a mediolateral gradient. The habenula and the ventral anterior nucleus were among the most weakly innervated nuclei. In the latter nucleus, as well as in more densely innervated nuclei, thin varicose fibers formed numerous pericellular contacts on cell bodies and proximal dendrites of thalamic neurons. The 5-HT innervation of the lateral nuclear group as well as that of the medial and lateral geniculate nuclei ranged from very weak to dense. The mediodorsal nucleus displayed a highly heterogeneous 5-HT innervation that varied from weak in its central portion to moderate or dense in its medial and lateral borders. A moderate 5-HT innervation was observed in the anterior nuclear group. The surprisingly dense and heterogeneous 5-HT innervation of the thalamus noted in the present study suggests that serotonin may be involved in several specific functions of the thalamus in primates.     

Topographic relations of cholinergic and noradrenergic neurons in the feline pontomesencephalic tegmentum: an immunohistochemical study

The immunohistochemical localization of the neurotransmitter synthesizing enzymes choline acetyltransferase, tyrosine hydroxylase and dopamine-beta-hydroxylase was examined in the feline pontomesencephalic tegmentum. Examination of adjacent sections stained for either choline acetyltransferase, tyrosine hydroxylase or dopamine-beta-hydroxylase immunoreactivity, as well as individual sections doubly stained for both choline acetyltransferase and tyrosine hydroxylase immunoreactivity, unequivocally demonstrated that noradrenergic and cholinergic neurons were extensively intermingled in the brainstem tegmentum of the cat. This contrasts with the situation in various other species, where neurons utilizing these two neurotransmitters are discretely localized in distinct nuclei. Furthermore, the present studies demonstrate the existence of two types of choline acetyltransferase immunoreactive neurons in the feline tegmentum: the magnocellular neurons of the pedunculopontine and laterodorsal tegmental nuclei which stain histochemically for NADPH diaphorase, plus a population of small spindle-shaped neurons in the medial and lateral parabrachial nuclei which do not stain positively for NADPH diaphorase. The data are discussed with respect to several influential hypotheses of sleep cycle control.     

Origin of cingulate cortex cholinergic innervation in the rat

The relative contribution of the n. diagonal band and thalamic nuclei to the cholinergic innervation of the cingulate cortex was examined. Lesions were placed in the n. diagonal band, anterior thalamus, and medial thalamus of rats, and changes in choline acetyltransferase in discrete regions of the cingulate cortex were determined. The n. diagonal band lesion produced a large decrease in choline acetyltransferase activity while the thalamic lesions produced no significant change in activity.     

Retrograde double-labeling study of the mammillothalamic and the mammillotegmental projections in the rat

Collateral axonal branching from the medial or lateral mammillary nuclei to the anterior thalamus, Gudden's tegmental nuclei, the nucleus reticularis tegmenti pontis, and the medial pontine nucleus was studied using the fluorescent retrograde double-labeling method. One day after injection of Fast Blue into the anterior thalamic nuclei or Gudden's tegmental nuclei, Nuclear Yellow was injected into Gudden's tegmental nuclei or the nucleus reticularis tegmenti pontis and the medial pontine nucleus. Following 1 day survival, single- and double-labeled neurons were examined in the mammillary nuclei. The lateral mammillary nucleus contains neurons whose collateral fibers project to both the dorsal tegmental nucleus of Gudden and the ipsilateral or contralateral anterodorsal thalamic nucleus, to both the medial pontine nucleus and the anterodorsal thalamic nucleus, and to both the dorsal tegmental nucleus of Gudden and the medial pontine nucleus. The pars medianus and pars medialis of the medial mammillary nucleus contain neurons whose collateral fibers project to both the anteromedial thalamic nucleus and the ventral tegmental nucleus of Gudden, to both the anteromedial thalamic nucleus and the medial part of the nucleus reticularis tegmenti pontis, and to both the ventral tegmental nucleus of Gudden and the medial part of the nucleus reticularis tegmenti pontis. The dorsal half of the pars posterior of the medial mammillary nucleus contains a few neurons whose collateral fibers project to both the anteromedial thalamic nucleus and the rostral part of the ventral tegmental nucleus of Gudden, and to both the caudal part of the anteroventral thalamic nucleus and the rostral part of the ventral tegmental nucleus of Gudden, while the pars lateralis of the medial mammillary nucleus contains no double-labeled neurons and projects only to the anteroventral thalamic nucleus.     

Neurochemical and histochemical studies of the effect of a lesion of the nucleus cuneiformis on the cholinergic innervation of discrete areas of the rat brain

The innervation sites of the dorsal tegmental acetylcholinesterase (AChE)-containing pathway were examined in rats by combining histochemical and biochemical techniques. A lesion was placed in the nucleus cuneiformis (midbrain reticular formation) and brains were examined after 4 days survival for changes in AChE staining and choline acetyltransferase (ChAT) activity in discrete brain areas. An ipsilateral projection appears to exist to the anterior thalamic nuclei, lateral portion of the medial thalamic nucleus, parafascicular nucleus, pretectal nucleus, posterior thalamic nucleus, and deep layers of the superior colliculus. A possible bilateral innervation to the reticular nucleus of the thalamus and the dorsal and ventral lateral geniculates was found. The parallel use of AChE histochemistry and measurements of ChAT activity in discrete nuclei will be useful for future evaluation of cholinergic pathways.     

Distribution of choline acetyltransferase and acetylcholinesterase in vocal control nuclei of the budgerigar (Melopsittacus undulatus)

The present study used histochemical methods to map the distributions of choline acetyl transferase (ChAT) and acetylcholinesterase (AChE) in the vocal control nuclei of a psittacine, the budgerigar (Melopsittacus undulatus). The distributions of ChAT and AChE in budgerigars appeared similar to that in oscine songbirds despite evidence that these systems have evolved independently. The magnicellular nucleus of the lobus parolfactorius in budgerigars, like the area X in songbirds, contained many ChAT labeled somata, fibers, and varicosities and stained densely for AChE. In contrast, the robust nucleus of the archistriatum (RA) and the supralaminar area of the frontal neostriatum in budgerigars, like the RA and the magnicellular nucleus of the neostriatum (MAN) in songbirds, respectively, contained few or no ChAT labeled somata, fibers, and varicosities and stained lightly for AChE. The central nucleus of the lateral neostriatum in budgerigars, like the higher vocal center (HVC) in songbirds, contained no ChAT labeled somata, moderate densities of ChAT labeled fibers and varicosities, and moderate levels of AChE staining. Two nuclei, the oval nucleus of the hyperstriatum ventrale (HVo) and the oval nucleus of the anterior neostriatum (NAo), contained no ChAT labeled somata, dense ChAT labeled fibers and varicosities, and moderate to high levels of AChE staining. The HVo and the NAo have no counterparts in songbirds but may be important vocal control nuclei in the budgerigar. Cholinergic enzymes are also described in other regions which may be involved in budgerigar vocal behavior, including the basal forebrain, the torus semicircularis, and the hypoglossal nuclei (nXII). © 1996 Wiley-Liss, Inc.     

Auditory thalamocortical pathways defined in monkeys by calcium-binding protein immunoreactivity

This study investigated differentiation of Macaca fuscata auditory thalamus into chemically defined nuclei forming relays to auditory cortical areas. The thalamus was stained immunocytochemically for parvalbumin and 28 kDa calbindin in normals and in brains in which retrogradely transported tracers were injected into middle layers of auditory cortical areas or applied to the cortical surface. Parvalbumin- and calbindin-immunoreactive cells show a complementary distribution in ventral, anterodorsal, post and magriocellular medial geniculate nuclei. The ventral nucleus has a high density of parvalbumin cells and few calbindin cells, and the anterodorsal nucleus has a high density of parvalbumin cells and moderate numbers of calbindin cells. Both nuclei have a dense parvalbumin-immunoreactive neuropil formed by terminations of fibers ascending in the brachium of the inferior colliculus. The posterodorsal nucleus has approxi mately equal proportions of parvalbumin and calbindin cells; neuropil staining is weak but contains terminations of calbindin-immunoreactive fibers ascending in the midbrain tegmenturn. The magnocellular nucleus contains domains of parvalbumin and calbindin cells. Parvalbumin cells in the ventral nucleus project to a central core of auditory cortex with densest parvalbumin immunoreactivity. Those in anterodorsal and posterodorsal nuclei project to surrounding auditory fields with less dense parvalbumin immunoreactivity; those in the magnocellular nucleus project widely to auditory and other fields. Injections of middle cortical layers label a large majority of parvalbumin cells in the ventral, anterodorsal, or posterodorsal nuclei and in the magnocellular nucleus. Superficial deposits label calbindin cells only, usually in more than one nucleus, implying a widespread projection system. © Wiley-Liss, Inc.     

Distribution of neuromedin U-like immunoreactivity in the central nervous system of Rana esculenta

The distribution of perikarya and nerve fibers containing neuromedin U-like immunoreactivity in the brain of Rana esculenta was determined with an antiserum directed toward the carboxyl terminus of the peptide. In the telencephalon, immunoreactive perikarya were found in the olfactory bulb, the medial septum, and the diagonal band. In the diencephalon, labeled perikarya were detected in the anterior and posterior preoptic areas, the dorsal nucleus of the hypothalamus, the caudal part of the infundibulum, and the posterior tuberculum. In the mesencephalon, immunoreactive cell bodies were found only in the laminar nucleus of the torus semicircularis and the anterodorsal tegmental nucleus. In the rhombencephalon, labeled perikarya were detected in the secondary visceral nucleus, the cerebellar nucleus, the central gray, and the nucleus of the solitary tract. Immunoreactive nerve fibers were observed in all areas of the brain that contained labeled perikarya. The densest accumulations were found in the nucleus accumbens; the dorsal part of the lateral septum; the periventricular region of the ventral thalamus; the lateral part of the infundibulum; the anterodorsal, anteroventral, posterodorsal, and posteroventral tegmental nuclei; and the periaqueductal region of the tegmentum. The distribution of neuromedin U-like immunoreactivity in the frog brain was substantially different from the distribution described for the rodent brain. © 1996 Wiley-Liss, Inc.     

Substance P and cholecystokinin-like immunoreactive varicosities in somatosensory and autonomic regions of the rat spinal cord: a quantitative study of coexistence

Substance P- and cholecystokinin-like immunoreactivities have been shown to coexist in some, but not all, dorsal root ganglion cell bodies of the rat. Quantitative immunofluorescence techniques were used in the present study to describe densities of substance P- and cholecystokinin-like immunoreactive varicosities in several spinal cord nuclei. By combining simultaneous immunofluorescent techniques on one tissue section with computerized image processing, coexistence of substance P- and cholecystokinin-like immunoreactivity in varicosities was also quantified. By comparing spinal cord regions between normal and unilateral dorsal rhizotomy affected animals, densities of substance P- and cholecystokinin-like immunoreactive varicosities associated with primary afferent neurons were established. To determine the densities of immunoreactive varicosities that were related to unmyelinated primary afferent fibers, data were compared between normal animals and those treated neonatally with capsaicin. Four major observations were made: (1) Substance P- and cholecystokinin-like immunoreactivity coexist in populations of varicosities in sensory and autonomic regions of rat spinal segment L6. (2) Within the superficial laminae of the dorsal horn, varicosities containing both substance P- and cholecystokinin-like immunoreactivity and cholecystokinin-like immunoreactivity alone are of primary afferent neuron origin, but those containing only substance P-like immunoreactivity are most likely of spinal or descending neuronal origin. (3) Capsaicin-insensitive cholecystokinin-like immunoreactive varicosities were present predominantly in lamina I. These data suggest some cholecystokinin-like immunoreactive varicosities are associated with myelinated primary afferent neurons. (4) Primary afferent fibers containing substance P- and cholecystokinin-like immunoreactivity project to intermediate gray regions of the rat spinal cord. A large proportion of these fibers are capsaicin sensitive, suggesting that they are unmyelinated.     

Immunocytochemical localization of choline acetyltransferase-like immunoreactivity in the guinea pig cochlea

The immunocytochemical localization of the enzyme choline acetyltransferase (ChAT) was examined in the guinea pig organ of Corti to determine if both lateral and medial systems of efferents would show immunoreactive labeling for this specific enzyme marker of cholinergic neurons. Cochleae were also examined after lesion of efferents to determine if ChAT-like immunoreactivity is confined to efferents. ChAT-like immunoreactivity was seen in the inner spiral bundle, tunnel spiral bundle and by the bases of inner hair cells corresponding to the lateral system of efferents. ChAT-like immunoreactivity was also seen in crossing fibers and puncta at the bases and by the nuclei of outer hair cells corresponding to the medial system of efferents. With the use of video enhanced contrast microscopy more than 9 ChAT-like immunoreactive puncta at the bases of outer hair cells could be resolved. In cochleae examined 6 weeks after ipsilateral lesion of efferents, no ChAT-like immunoreactivity was observed. These results add strong evidence that acetylcholine is a transmitter of both the medial and lateral systems of efferents.     

Projection from the external pallidum to the reticular thalamic nucleus in the squirrel monkey

Small injections of biocytin in the external segment of the pallidum (GPe) of the squirrel monkey (Saimiri sciureus) led to anterograde labeling of fibers in the thalamic reticular nucleus (NRT). These fibers reached NRT by coursing along the ventral tip of the internal capsule or by directly piercing the internal capsule more dorsally. They arborized profusely within the entire rostrocaudal extent of the nucleus. Within NRT, biocytin-labeled fibers were long, slightly varicosed, and emitted numerous short collaterals whose terminal portions consisted of clusters of large varicosities. Some of these varicosities were closely apposed to cell bodies and proximal dendrites of NRT neurons. Small injections of wheat germ-agglutinated horseradish peroxidase in the rostral pole of NRT led to retrograde cell labeling within the entire rostrocaudal extent of GPe. These retrogradely-labeled cells did not display immunoreactivity for choline acetyltransferase. Hence, beside the well-established projection from the internal pallidum to the thalamus, our findings support the existence of another pallidothalamic projection whereby GPe neurons could exert a powerful influence upon the thalamocortical neurons via a relay in NRT.     

Postnatal changes in the density and distribution of neurotensin-like immunoreactive fibers in the mediodorsal nucleus of the thalamus in the rat.

A previous report (Inagaki et al., Brain Res. 260:143-146, '83) suggested that the peptide neurotensin is contained in neurons of the piriform cortex that project to the mediodorsal thalamic nucleus (MD) in young rats. To confirm this, we have studied the distribution of neurotensin-like immunoreactive (NTIR) fibers in MD during development, using three antisera directed at different parts of the neurotensin molecule (Emson et al., J. Neurochem. 38:992-999, '82). In adult rats, NTIR fibers in MD are sparse. They are located mostly at the medial edge of MD and in the adjacent midline thalamic nuclei, with a few poorly stained NTIR fibers in the central part of MD. In contrast, during the first postnatal week, both the medial and central portions of MD stain heavily for neurotensin. The density of NTIR fibers in MD then progressively decreases until the density typical of adult rats is reached, at about 5 weeks. Changes in the distribution of NTIR fibers in MD also occur. In 7-day-old rats, the patches of NTIR fibers in the medial and central parts of MD are contiguous, but by 10 days a sparsely immunoreactive zone forms between them. With maturation, this zone enlarges as the density of neurotensin staining decreases, until the medial contingent of NTIR fibers reaches its adult position at the medial edge of MD. From a comparison of the distribution of NTIR cells with that of cells that can be retrogradely labeled from MD or the midline thalamus, the probable source of the NTIR fibers to the central part of MD is in the deep layer of the piriform cortex, while the NTIR fibers to the medial edge of MD and the midline nuclei may arise from the preoptic region and the medial amygdala. In neonatal rats, neurons are found in the piriform cortex, the preoptic region, and the medial amygdala, which can be double-labeled both for neurotensin and with a retrograde tracer injected into MD and the midline thalamus. Projections of the preoptic region to the thalamus have a distribution similar to that of the medial population of NTIR fibers, whereas the distribution of piriform cortical afferents in central MD matches the central patch of NTIR fibers.     

Distribution of prosaposin-like immunoreactivity in rat brain

Prosaposin is the precursor for saposins A, B, C, and D, which are small lysosomal proteins required for the hydrolysis of sphingolipids by specific lysosomal hydrolases. With a monospecific anti-saposin C antibody, which cross-reacts with prosaposin but not with saposin A, B, or D, the present immunoblot experiments showed that the rat brain expresses an unprocessed ～ 72 kDa protein (possibly prosaposin) and little saposin C. Regional analysis demonstrated that prosaposin is abundant in the brainstem, hypothalamus, cerebellum, striatum, and hippocampus, and less abundant in the cerebral cortex. Consistent with this finding, prosaposin-like immunoreactive neurons and fibers as revealed by immunohistochemistry were observed frequently in subcortical regions. The medial septum, diagonal bands, basal nucleus of Meynert, ventral striatum, medial habenular nucleus, and motor nuclei of cranial nerve had significant numbers of immunoreactive neurons. There were also nerve fibers with prosaposin-like immunoreactivity in several projection fields of the above nuclei. Other brain areas that contained prosaposin-like immunoreactive neurons and/or processes were: several brain nuclei (nucleus caudate putamen, globus pallidus, substantia nigra, red nucleus) constituting the so-called extrapyramidal system, reticular thalamic nucleus, entopeduncular nucleus, mammillary nuclei, auditory relay nuclei, cerebellum, sensory cranial nerve nuclei, and the reticular formation. The distribution pattern of prosaposin is apparently different from that of other neuroactive substances so far examined, and thus prosaposin may be involved in novel central events. © 1993 Wiley-Liss, Inc.     

Plasticity of Galaninergic Fibers Following Neurotoxic Damage within the Rat Basal Forebrain: Initial Observations

Galanin immunoreactive fibers hypertrophy and hyperinnervate remaining cholinergic basal forebrain neurons within the septum–diagonal band complex in Alzheimer's disease. The present investigation determined whether a similar hyperinnervation of galanin immunoreactive fibers occurs following intraparenchymal injections of ibotenic acid within the cholinergic medial septum or diagonal band nucleus in young adult rats. Sections through the medial septum and the diagonal band were either concurrently immunostained for galanin and the low-affinity p75 neurotrophin receptor (an excellent marker of cholinergic basal forebrain neurons) or single stained for choline acetyltransferase. Following chemical lesion, an increase in the density of galanin immunoreactivity was seen within the medial septum on the lesion, as opposed to the contralateral control side. In contrast, within diagonal band-lesioned animals, the increase in galanin immunoreactivity was low to moderate. In either lesion paradigm we did not observe hyperinnervation of remaining cholinergic basal forebrain neurons. In fact, there was no correlation between the galanin hypertrophy and the amount of cholinergic cell loss. We hypothesize that galanin hyperinnervation within the cholinergic basal forebrain may provide a protective effect by down-regulating acetylcholine release following brain insult.     

Light and electron microscopic study of m2 muscarinic acetylcholine receptor in the basal forebrain of the rat

The m2 muscarinic acetylcholine receptor gene is expressed at high levels in basal forbrain, but the paucity of information about localization of the encoded receptor protein has limited the understanding of cellular and subcellular mechanisms involved in cholinergic actions in this region. The present study sought to determine the cellular localization of m2 protein, its relationship to cholinergic neurons, and its pre-and postsynaptic distribution in the rat medial septum-diagonal band complex using immunocytochemistry with polyclonal rabbit antibodies and a newly developed rat monoclonal antibody specific to the m2 receptor. Light microscopic colocalization studies demonstrated that m2 was present in a subset of choline acetyltransferase immunoreactive neurons, in choline acetyltransferase-negative neurons, and in more neuropil elements than was choline acetyltransferase. Intraventricular injections of 192 IgG-saporin, an immunotoxin directed to the low-affinity nerve growth factor receptor, resulted in depletion of choline acetyltransferase-immunoreactive neurons in the medial septum-diagonal band complex, whereas m2 immunoreactivity in neurons and in the neuropil was unchanged. By electron microscopy, m2 receptor in medial septum-diagonal band complex was localized to the plasmalemma of a small population of small to medium-sized neurons, and it was also found in dendrites, axons, and axon terminals in the neuorpil Neurons expressing m2 immunoreactivity recived synaptic contacts from unlabelled axon terminals. A small distinct subpopulation of large neurons, unlabelled by m2 immunoreactivity, received synaptic contacts from m2-immunoreactive terminals. Thus, m2 receptor is situated to mediate the local effects of acetylcholine on basal forebrain cholinergic and noncholinergic neurons and, also, at both pre-and postsynaptic sites. © 1995 Willy-Liss, Inc.