Development of the rat septohippocampal projection: Tracing with DiI and electron microscopy of identified growth cones

The factors determining the development of specific fiber tracts in the central nervous system as well as the interactions of growth cones with the surrounding micromilieu are largely unknown. Here we investigated the ontogenetic development of the septohippocampal projection in the rat with the lipophilic carbocyanine dye DiI which is transported anterogradely and retrogradely in neurons and can be applied to fixed embryonic tissue. Photoconversion of anterogradely labeled fibers allowed us to study individual growth cones by electron microscopy. The first axons originating from the septal complex were found in the hippocampus as early as on embryonic day (ED) 19, reaching the fimbrial pole of the hippocampus on ED 18. However, on ED 17 we consistently found retrogradely labeled cells in the hippocampus, indicating that the development of the hippocamposeptal projection precedes that of the septohippocampal projection. On ED 19, the majority of the axons directed toward the hippocampal formation passed the hippocampus and grew further into the subicular complex and entorhinal cortex. These axons gave off collaterals that invaded the hippocampus proper. A fairly adult pattern of the septohippocampal projection was reached on postnatal day 10, although many growth cones were still found. A comparative analysis of individual growth cones found in the fimbria and the hippocampus proper revealed no striking differences in their morphology. Electron microscopic analysis showed that growth cones in the fimbria were mainly contacted by other axons, whereas growth cones in the hippocampus had contact with all available elements. This may indicate that growing septohippocampal fibers are guided by axons of the earlier formed hippocamposeptal projection. In the hippocampus proper, other cues, probably derived from the target itself, may guide the septhippocampal axons to their appropriate target cells. © 1993 Wiley-Liss, Inc.     

Nerve growth factor receptor immunoreactivity in the rat suprachiasmatic nucleus

Using a monoclonal antibody, nerve growth factor receptor has been immunohistochemically identified within the suprachiasmatic nucleus of adult rats. Labelling was most intense in the ventral, lateral and caudal portions of the nucleus and appeared primarily associated with fibers and terminals. These were among the most intensely labelled fibers and terminals in the forebrain.     

Gamma-aminobutyric acid-immunoreactivity in the rat hippocampus. A light and electron microscopic study with anti-GABA antibodies

The distribution of GABA-immunoreactive neurons and axonal varicosities was investigated in the hippocampal region of the rat brain by means of an indirect peroxidase immunocytochemical method with recently developed anti-GABA antibodies. The immunolabeling was found to be restricted to nervous structures: neuronal cell bodies, dendrites and axon terminals. Myelinated axons showing GABA-immunoreactivity were also observed. GABA-immunoreactive neurons were found in great number in the stratum pyramidale, the superficial part of the stratum oriens and the deep part of the stratum radiatum in the Ammon's horn. Less were found in the other regions; rare labeled cells were observed in the superficial part of the stratum radiatum and the middle part of the stratum oriens. The dentate gyrus exhibited numerous labeled cells in the granular layer, few in the hilus, rare in the molecular later. A high density of GABA-immunoreactive terminals was found at the limit of the stratum oriens with the alveus, in the stratum pyramidale and in the stratum lacunosum. A lower density of labeled fibers was observed in the other areas. The somata and proximal dendrites of pyramidal and granular cells were encompassed by characteristics pericellular arrangements of GABA-immunoreactive varicosities. Ultrastructural observations revealed a diffuse immunoreaction product spread over the cytoplasm and the nucleus without specific relationship with the organelles, and immunoreactive aggregates in the cytoplasm. Labeled dendrites often showed enlargements displaying the immunoreaction whereas thinner segments were devoid of it. They received numerous asymmetrical synapses from unlabeled axon terminals. GABA-immunoreactive terminals were filled with small clear vesicles with immunopositive membranes and were observed in symmetrical contact with somata and dendrites.     

Gap junction protein in rat hippocampus: correlative light and electron microscope immunohistochemical localization

Immunohistochemical techniques and an affinity-purified antibody directed against the 27-kD gap-junctional protein (GJP) from rat liver were used to determine the ultrastructural localization of GJP in the rat hippocampus. At the light microscope level, dense GJP immunoreactivity having a stringlike appearance was seen in a very small percentage of medium-sized neuronal somata located in the stratum pyramidale, and diffuse immunostaining was seen in many small cell bodies in the stratum pyramidale, stratum oriens, and the alveus. Abundant GJP-immunoreactive (GJP-IR) varicose fibers were observed in the strata pyramidale, radiatum, and oriens but were less concentrated in the alveus. Numerous punctate GJP-IR elements were observed in all hippocampal layers. Upon EM analysis, GJP-IR neuronal somata in the stratum pyramidale were found to be, without exception, nonpyramidal neurons as judged by such distinguishing features as their fusiform perikarya, indented nucleus, and well-developed rough endoplasmic reticulum (RER). Immunostaining within these cells was largely localized to the Golgi apparatus and associated vesicular components. Small, diffusely GJP-IR cells were identified ultrastructurally as protoplasmic and fibrous astrocytes. Immunostaining within these cells was localized to the Golgi apparatus, RER, and small, ribosomelike bodies 15-25 nm in diameter. Among neuronal processes GJP immunoreactivity was found within dendrites, axons, and axonal terminals. The latter structures contained numerous GJP-IR vesicles having an average diameter of about 40 nm. A frequent observation indicating some degree of specificity of the anti-GJP antibody employed here was immunostaining of typical gap junctions between dendrites and, more commonly, between processes of glial cells. Occasionally, however, GJP-IR dendrodendritic, axodendritic, and axoaxonic contacts were found that could be considered, at best, as being gap-junction-like (gj-L). In these cases, asymmetric immunostaining of adjacent plasma membranes forming gj-L structures was not uncommon. These results confirm the existence of gap junctions between dendrites in the rat hippocampus and demonstrate that GJP immunoreactivity on cytoplasmic membranes is restricted either to typical neuronal and glial gap junctions or to gj-L structures at circumscribed sites of contact between various types of neuronal elements where GJP may contribute to a novel mechanism of neural communication.     

Ultrastructural localization of neuropeptide Y-like immunoreactivity in the rat hippocampal formation.

Neuropeptide Y (NPY) has been implicated in the modulation of hippocampal neuronal activity and in the pathophysiology of several neurological disorders involving the hippocampal formation. Thus, this study examines the light and electron microscopic immunoperoxidase labeling of a rabbit polyclonal antibody against porcine NPY in single sections through each lamina of the CA1 and CA3 regions of the hippocampus and the dentate gyrus (DG) of normal adult rats. By light microscopy, the majority of perikarya with intense NPY-like immunoreactivity (NPY-LI) were located in stratum oriens of CA1 and CA3 of the hippocampus and in the hilus of the DG. Fine varicose processes with NPY-LI were found in all layers of the hippocampal formation, but were densest in the outer third of the molecular layer of the DG. The density of NPY-labeling was greater in the ventral portion of the hippocampal formation. By electron microscopy, most NPY-containing perikarya in all three hippocampal regions were: small (8-12 microns) or medium-sized (12-18 microns) and elongated; or medium-sized and round. A dense accumulation of NPY-LI was commonly observed within the individual saccules of Golgi complexes and some rough endoplasmic reticulum in the cytoplasm. Perikarya and dendrites with NPY-LI usually were directly apposed to other neuronal processes (mostly terminals) and lacked astrocytic appositions. The majority of terminals in contact with NPY immunoreactive neurons were unlabeled and synapsed with the shafts of large and small dendrites. In CA1 and CA3 of the hippocampus, the types of synapses formed by the unlabeled terminals were not significantly different; however, more asymmetric synapses than symmetric synapses were formed by the unlabeled terminals on the shafts of small NPY-labeled dendrites in the DG. The terminals with NPY-LI (0.25-1.2 microns) contained many small, clear vesicles and 0-2 large, dense-core vesicles. The types of synapses (i.e., asymmetric and symmetric) and distribution of NPY-labeled terminals on the targets were remarkably similar in each lamina of the hippocampal subregions. The NPY-labeled terminals usually synapsed with one unlabeled perikaryon or dendrite. However, others synapsed either (1) with two unlabeled perikarya or dendrites simultaneously or (2) with one NPY-containing perikaryon or dendrite. Most of the terminals with NPY-LI formed symmetric junctions with the shafts of small (distal) dendrites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nerve growth factor receptor immunoreactivity in the new world monkey (Cebus apella) and human cerebellum.

The present study used the NGFR-5 monoclonal antibody raised against human nerve growth factor receptor (NGFR) to determine the extent of NGFR immunoreactivity within the embryonic and young adult Cebus apella cerebellum as well as the human cerebellum. Immunohistochemically processed tissue revealed NGFR expressing Purkinje cell somata, axons, and dendrites, the latter being observed within the molecular layer of both adult species. Within all regions of the cerebellum we observed both darkly and lightly immunostained Purkinje cells. The proximal axons of these cells, which were visualized for short distances within the granular cell layer, appeared to contain bulbous aggregates of reaction product. In sagittal sections, the full extent of the Purkinje cell dendritic tree was observed in the more lightly stained portions of the cerebellum. In situ hybridization experiments revealed NGFR mRNA within Purkinje cells in a pattern similar to that seen with immunohistochemistry. The distribution of NGFR immunoreactivity within the cerebellum exhibits a general topographic organization with the heaviest and most consistent staining occurring within the archi- and neocerebellum and weaker staining within the paleocerebellum. In fetal Cebus monkey cerebellum obtained at gestational day 50 and 70, NGFR immunoreactivity was observed as a band composed of developing Purkinje cell neurites. These profiles were seen in the paleo- and neocerebellum, but not the archicerebellum. The present investigation is the first demonstration of NGFR immunoreactive profiles in the adult monkey and human cerebellum. These findings suggest that nerve growth factor may influence locomotor and vestibular behaviors that are mediated by cerebellar circuity. The precise mode of action for the NGF/NGFR system within the cerebellum remains to be determined.     

Projection from the nucleus reuniens thalami to the hippocampal region: light and electron microscopic tracing study in the rat with the anterograde tracer Phaseolus vulgaris-leucoagglutinin

In order to study the morphological substrate of possible thalamic influence on the cells of origin and area of termination of the projection from the entorhinal cortex to the hippocampal formation, we examined the pathways, terminal distribution, and ultrastructure of the innervation of the hippocampal formation and parahippocampal region by the nucleus reuniens of the thalamus (NRT). We employed anterograde tracing with Phaseolus vulgaris-leucoagglutinin (PHA-L). Injections of PHA-L in the NRT produce fiber and terminal labeling in the stratum lacunosum-moleculare of field CA1 of the hippocampus, the molecular layer of the subiculum, layers I and III/IV of the dorsal subdivision of the lateral entorhinal area (DLEA), and layers I and III-VI of the ventral lateral (VLEA) and medial (MEA) divisions of the entorhinal cortex. Terminal labeling is most dense in the stratum lacunosum-moleculare of field CA1, the molecular layer of the ventral part of the subiculum, MEA, and layer I of the perirhinal cortex. In layer I of the caudal part of DLEA and in MEA, terminal labeling is present in clusters. Injections in the rostral half of the NRT produce the same distribution in the hippocampal region as those in the caudal half of the NRT, although the projections from the rostral half of the NRT are much stronger. A topographical organization is present in the projections from the head of the NRT, so that the dorsal part projects predominantly to dorsal parts of field CA1 and the subiculum and to lateral parts of the entorhinal cortex, whereas the ventral part projects in greatest volume to ventral parts of field CA1 and the subiculum and to medial parts of the entorhinal cortex. The distribution of the reuniens fibers coursing in the cingulate bundle was determined by comparing cases with and without transections of this bundle. The fibers carried by the cingulate bundle exclusively innervate field CA1 of the hippocampus, the dorsal part of the subiculum, and the presubiculum and parasubiculum. They participate in the innervation of the ventral part of the subiculum and MEA. Electron microscopy was used to visualize the axon terminals of PHA-L-labeled reuniens fibers. These terminals possess spherical synaptic vesicles and form asymmetric synaptic contacts with dendritic spines or with thin shafts of spinous dendrites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Serotonin-containing terminals synapse on septohippocampal neurons in the rat

Serotonin [5-hydroxytryptamine (5-HT)] is thought to be involved in mnemonic functions and dysfunctions possibly by directly contacting neurons in the medicl septal and diagonal band nuclei (i.e., the septal complex) that project to the hippocampal formation. However, there is no cellular substrate for this modulation. Thus, we examined the ultrastructure and synaptic association of 5-HT-containing terminals in relation to septohippocampal neurons in the septal complex of the rat brain. Projection neurons were identified by retrograde transport of wheat germ agglutinated apo-horseradish perodidase conjugated to colloidal gold particles (WANG) following an injection into the ventral hippocampal formation of uneashetized adult rats. After a 1 day survival, setions through the septal complex were labeled with antibodies to 5-HT immunoreactivity (5-HT-I) were observed in close proximity to neurons containing retogardely transported WAHG. By electron microscopy, 5-HT-I immunoreacitity (5-HT-I) were observed in close proximity to neurons containing retrogadely transported WAHG. By electron microscopy, 5-HT-I was found exclusively in axons and axon terminals. Axons were primarily unmyelinated. Terminals with 5-HT-I were 0.35–1.2 μm in diameter and contained numerous small, clear vesicles and 0–4 large, dense-core vesicles. The 5-HT-labeled terminals: (1) contacted perikarya and dendrites (220 of 349); (2) were closely apposed to other terminals (25 of 349); or (3) had no neuronal contacts in the plane of section analyzed (104 of 349). The 5-HT-labeled terminals formed exclusively symmetric synases on perikarya; some of these perikarya as well as some large dendrites similarly contacted by the 5-HT-labeled terminals also contained WAHG affiliated with lysosomes and multivesicular and “sequestration” bodies in the cytoplasm. However, the majority of terminals with 5-HT-I formed contacts on the shafts of small unlabeled dendrites (69% of 220); most of these were characterized as either asymmetric synapses or appositions not separated by astrocytes in the plane of section analyzed. We conclude that 5-HT-containing terminals in the rat septal complex: (1) directly modulate septohippocampal and other neurons through symmetric (potentially inhibitory) synapses on soma and proximal dendrites; and (2) form primarily asymmetric (potentially excitatory) synapses with distal (small) dendrites from neurons of unidentified origin. These findings suggest that serotonin may affect learning and memory through modulation of septal efferents to the hippocampal formation and may have direct relevance to the neuropathological basis for Alzheimer's disease. © 1993 Wiley-Liss, Inc.     

Ultrastructural localization of tyrosine hydroxylase-like immunoreactivity in the rat hippocampal formation

The light and electron microscopic localization of antigenic sites for a polyclonal antiserum directed against the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH), was examined in the hippocampal formation of the rat brain with a double-bridged peroxidase-antiperoxidase method. By light microscopy, the majority of varicose processes with intense TH-like immunoreactivity (LI) were contained in the hilus of the dentate gyrus (DG) and strata radiatum and lacunosum-moleculare of the CA3 region of the hippocampus. Only a few immunoreactive fibers were observed in the molecular and granule cell layers of the DG, in strata oriens and pyramidale of CA3, and in all layers of CA1. Electron microscopy confirmed that these labeled processes were primarily axons and axon terminals. Terminals with TH-LI were 0.4-1.1 micron in diameter and contained many small clear vesicles and from 0 to 3 larger dense-core vesicles. The number and types of associations formed by terminals with TH-LI were remarkably similar in the DG and hippocampus proper despite known differences in intrinsic cells and function. In both regions, the majority of terminals with TH-LI formed junctions on small (distal dendrites (52% of 112 in the DG; 67% of 116 in CA3) and dendritic spines (30% in the DG; 18% in CA3) that were both asymmetric and symmetric. In the DG, axosomatic junctions (2% of 112) were symmetric and occurred exclusively on the perikarya of granule cells, whereas junctions on large (proximal) dendrites were more numerous (16%), exhibited symmetric as well as asymmetric membrane specializations, and were of both granule (molecular layer) and nongranule (hilus) cell origin. In CA3, synaptic contacts on perikarya (5% of 116) and large (proximal) dendrites (10%) of both pyramidal cell and nonpyramidal cell origin were few and all symmetric. The distribution and types of synaptic associations formed by terminals with TH-LI in the CA1 region paralleled that seen in the CA3 region. In both the dentate and hippocampus proper, 10% of the terminals with TH-LI were observed closely apposed to unlabeled terminals that formed asymmetric synapses with dendrites and dendritic spines. In rare instances, TH-immunoreactive terminals were found in close association with the basement membrane of blood vessels, astrocytic processes, or with other unlabeled terminals not forming recognizable junctions. In addition TH-LI was occasionally detected within the cytoplasm of a minority of astrocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructural localization of somatostatin-like immunoreactivity in the rat dentate gyrus

Neurons containing somatostatin (SOM) are enriched in the dentate gyrus. We sought to establish the ultrastructural localization of this peptide in the dentate gyrus of the rat brain with a double-bridged peroxidase-antiperoxidase (PAP) method localizing antisera directed against somatostatin (SOM)-28 and SOM-28. Initial light microscopic observations confirmed that the majority of perikarya and thick varicose processes with intense SOM-like immunoreactivity (SOM-LI) were observed in the hilus. Fine varicose processes with SOM-LI were found throughout all layers of the dentate gyrus but were most intense in the outer third of the molecular layer (ML), where an occasional perikaryon with SOM-LI was seen. By electron microscopy, SOM-LI was found in neuronal perikarya, dendrites, axons, and axon terminals. Two types of SOM-containing perikarya were observed. The first type was small (6-10 microns), round or avoid, and had a labeled cytoplasma with abundant Golgi complexes and a dense accumulation of PAP-reaction product. The second type of perikarya was larger (11-16 microns) and had a more abundant cytoplasm than the first type, but the Golgi complexes did not appear labeled. Most (96% of 374) of the synapses on the SOM-labeled perikarya and dendrites were from terminals without SOM-LI which formed nearly equal proportions of asymmetric and symmetric junctions. The remainder of the presynaptic terminals contained SOM-LI and made primarily symmetric synapses. Synaptic junctions from both unlabeled and labeled terminals were primarily on the shafts of the small (0.5-1.5 microns) SOM-immunoreactive dendrites. The terminals with SOM-LI (0.25-1.3 microns) contained many small, clear vesicles and from zero to four large dense-core vesicles. Terminals with SOM-LI were associated 1) with one unlabeled perikaryon or dendrite (49% of 215 in the hilus; 76% of 326 in the ML); 2) with two unlabeled perikarya or dendrites simultaneously (5% hilus; 4% ML); and 3) with one SOM-containing perikaryon or dendrite (6% hilus; 3% ML). In all three types of associations, synaptic contacts on perikarya were few while the majority were with small (distal) dendrites. Moreover, most of the terminals with SOM-LI formed symmetric junctions or lacked membrane specializations but were without any apparent glial intervention in the plane of section analyzed. The remaining SOM-labeled terminals (40% hilus; 17% ML) were without any apparent synaptic relations. However, a few of these terminals were in direct apposition to other terminals, some of which were also SOM-immunoreactive.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructural localization of tyrosine hydroxylase immunoreactivity in the rat diagonal band of Broca.

The present study sought to establish the cellular basis for the catecholaminergic (i.e., noradrenaline and dopamine) modulation of neurons in the horizontal limb of the diagonal band of Broca (HDB) in the rat brain. The light and electron microscopic localization of antigenic sites for a polyclonal antibody directed against the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH), were examined in the HDB using a double-bridged, peroxidase-antiperoxidase method. By light microscopy, numerous punctate, varicose processes with intense TH-immunoreactivity (TH-I) were detected in the HDB. Additionally, a few small, bipolar, or multipolar TH-immunoreactive neurons were observed. Ultrastructural analysis of single sections revealed that the TH-labeled processes were axons and axon terminals. Axons (n = 134) with TH-I were primarily unmyelinated. Terminals with TH-I (n = 169) were 0.3-1.4 microns in diameter and contained many small, clear vesicles and 0-5 larger dense-core vesicles. The types of associations (i.e., asymmetric synapses, symmetric synapses, and appositions which lacked a membrane specialization in the plane of section analyzed) formed by the TH-labeled terminals were quantitatively evaluated. The TH-labeled terminals: (1) formed associations with unlabeled perikarya and dendrites (134 out of 169), (2) were closely apposed without glial intervention to unlabeled and TH-labeled terminals (11 out of 169), or (3) had no neuronal associations in the plane of section analyzed (24 out of 169). The relatively rare (n = 4) associations with unlabeled perikarya were mostly characterized by symmetric synaptic specializations. The majority of the TH-labeled terminals were associated with the shafts of small dendrites (66% of 134). Moreover, most of the associations on dendrites and dendritic spines were further characterized by asymmetric synaptic specializations; however, many were also appositions without any apparent glial intervention in the plane of section analyzed. Additionally, the TH-labeled terminals were often associated with only one dendrite, which, in the same plane of section, was sparsely innervated by other terminals. Astrocytic processes usually surrounded the portions of the terminals and dendrites not involved in the region of association. The TH-immunoreactive perikarya were small (7-12 microns), ovoid, and had an indented nucleus with some heterochromatin. Their scant cytoplasm contained mitochondria, Golgi complexes, and endoplasmic reticulum. A few immunoreactive dendrites, presumably derived from the local neurons, were also detected. Both TH-immunoreactive perikarya and dendrites were associated primarily with unlabeled terminals, although a few terminals with TH-I also contacted them.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of adrenomedullin-like immunoreactivity in the rat central nervous system by light and electron microscopy

Adrenomedullin is a peptide of marked vasodilator activity first isolated from human pheochromocytoma and subsequently demonstrated in other mammalian tissues. Using a polyclonal antiserum against human adrenomedullin-(22-52) amide and the avidin-biotin peroxidase complex technique, we have demonstrated by light and electron microscopy that adrenomedullin-like immunoreactivity is widely distributed in the rat central nervous system. Western blotting of extracts of different brain regions demonstrated the fully processed peptide as the major form in the cerebellum, whereas a 14-kDa molecular species and a small amount of the 18-kDa propeptide were present in other brain regions. Immunoreactive neurons and processes were found in multipolar neurons and pyramidal cells of layers IV-VI of the cerebral cortex and their apical processes, as well as in a large number of telencephalic, diencephalic, mesencephalic, pontine and medullary nuclei. Cerebellar Purkinje cells and mossy terminal nerve fibers as well as neurons of the cerebellar nuclei were immunostained, as were neurons in area 9 of the anterior horn of the spinal cord. Immunoreactivity was also found in some vascular endothelial cells and surrounding processes that probably originated from perivascular glial cells. Electron microscopy confirmed the light microscopy findings and showed the reaction product in relation to neurofilaments and the external membrane of small mitochondria. Immunoreactive terminal boutons were occasionally seen. The distribution of adrenomedullin-like immunoreactivity in the central nervous system suggests that it has a significant role in neuronal function as well as in the regulation of regional blood flow.     

Time course of the elevation of nerve growth factor (NGF) content in the hippocampus and septum following lesions of the septohippocampal pathway in rats

Axotomy of cholinergic neurons in the medial septum-diagonal band and degeneration of their terminals in the hippocampus resulting from fornix-fimbria lesions induce elevation of NGF content in these two brain regions. Postlesion levels of cholinergic neuron-specific ChAT activity in the septum suggest that endogenously produced NGF may, at least partly, promote survival of axotomized cholinergic neurons or induce ChAT activity in undamaged cells or both. These findings thus support the proposed trophic role for NGF in central cholinergic neurons.     

Nerve terminal glutamate immunoreactivity in the rat nucleus accumbens and ventral tegmental area after a short withdrawal from cocaine

Cocaine administration has been shown to alter glutamate transmission in numerous studies. Using quantitative electron microscopic immunogold labeling, our laboratory has previously reported that nerve terminal glutamate immunoreactivity is transiently altered following cocaine administration. The present study was undertaken to examine presynaptic nerve terminal glutamate immunoreactivity at shorter time points after withdrawal from cocaine. Animals received saline or cocaine for 7 days followed 3 days later by a cocaine or saline challenge. Most (>75%) cocaine-challenged animals had a heightened locomotor response to cocaine compared to the first day of cocaine and were considered behaviorally sensitized. One day after the challenge, glutamate immunogold-labeling was quantified in nerve terminals making asymmetrical synaptic contacts within the core and shell of the nucleus accumbens and ventral tegmental area. A single dose of cocaine did not alter the density of presynaptic nerve terminal glutamate immunoreactivity in the nucleus accumbens (NAc) or ventral tegmental area (VTA). The density of nerve terminal glutamate immunoreactivity in the shell, but not the core, was significantly increased in the animals receiving repeated cocaine. In the VTA the density of nerve terminal glutamate immunoreactivity did not change in the cocaine-sensitized group, but was significantly increased in the nonsensitized group. The finding that repeated cocaine treatment increased glutamate nerve terminal immunolabeling within the nucleus accumbens shell, but not the core, supports the hypothesis that glutamate synapses in the core and shell are differentially sensitive to repeated cocaine administration. Overall, our study does not support a role for changes in presynaptic glutamate in the development of behavioral sensitization.     

The localization of nerve growth factor-like immunoreactivity in the adult rat basal forebrain and hippocampal formation.

The role of nerve growth factor (NGF) as a target derived neurotrophic agent for specific cell populations in the peripheral nervous system has been well documented and much evidence suggests that NGF may serve a similar neurotrophic role in the CNS supporting the cholinergic neurons of the basal forebrain. Previous attempts to localize NGF by immunocytochemical methods, however, have not yielded evidence confirming the regional distribution expected based upon reported levels of extractable NGF. In the present study, affinity purified polyclonal antibodies to beta-NGF and a modified immunohistochemical protocol were used to demonstrate specific NGF-like immunoreactivity in the adult rat hippocampal formation and basal forebrain. In the hippocampal formation, NGF-like immunoreactivity was localized primarily within the hilus of the dentate gyrus and within stratum lucidum of the CA3 and CA2 hippocampal subfields. Staining appeared to be associated with cell processes and was similar to the reported distribution of mossy fibers suggesting that granule cells may either serve as a primary source of hippocampal NGF or that mossy fibers selectively accumulate NGF produced by other cell populations. In the basal forebrain, NGF-like immunoreactivity was localized within neuronal cell bodies of the medial septum, diagonal band, and nucleus basalis of Meynert and was further demonstrated to colocalize exclusively with LNGF-R positive neurons. These findings demonstrate the presence of an NGF-like antigen in association with cholinergic neurons of the basal forebrain and strongly support the hypothesis that NGF may serve as an endogenous trophic factor for this adult neuronal population.     

Substance P-like immunoreactivity in the lateral cervical nucleus of the owl monkey (Aotus trivirgatus): a comparison with the cat and rat

The location of substance P (SP) in the lateral cervical nucleus (LCN) of monkeys (Aotus trivirgatus), cats, and rats was investigated with immunohistochemical methods. Light microscopic analysis showed that SP-positive fibers and terminals are evenly distributed throughout the LCN of the monkey and rat, whereas the SP labeling in the LCN of the cat is concentrated in the medial part of the nucleus, with only very sparse labeling in the lateral part. Electron microscopic examination of the monkey LCN revealed the presence of SP-like immunoreactivity within terminal boutons and unmyelinated axons. The SP-positive boutons are in synaptic contact with dendrites and, occasionally, cell bodies; they contain densely packed, clear, round synaptic vesicles, as well as dense-core vesicles. The distribution of SP-like immunoreactivity in the LCN of monkeys, cats, and rats is similar to that of nociceptive-responsive neurons demonstrated in electrophysiological experiments. The possible role of the SP-containing fibers in the transmission of nociceptive information through the LCN is discussed.     

The subnuclear organization of the rat interpeduncular nucleus: a light and electron microscopic study

The subnuclear organization of rat interpeduncular nucleus (IPN) has been examined by light microscopy following staining with Nissl and Holmes methods, 3H-leucine autoradiography, acetylcholinesterase (AChE), and cytochrome oxidase histochemistry on plastic sections stained with toluidine blue, and by electron microscopy. Three unpaired and four paired subnuclei are recognized. The rostral subnucleus is heavily stained for AChE, which clearly delineates its borders. It is distinguished ultrastructurally by two types of synapses on dendrites, and two on perikarya. Of the former, one type is formed by presynaptic processes which contain spherical and dense-cored vesicles and make asymmetrical contacts. Dense-cored vesicles are observed in many of the postsynaptic dendrites. A second type has presynaptic processes containing small, pleomorphic vesicles which make symmetrical contacts. Synapses on perikarya are found in the rostral, central, intermediate, lateral, and interstitial subnuclei. The dorsal subnucleus is continuous with the serotonin-containing B8 cells. The central subnucleus is distinguished by longitudinally oriented medial habenular axons separating palisades of cell bodies. These axons, which also traverse the intermediate subnuclei, form en passant S synapses with small dendrites of the central subnucleus. The intermediate subnuclei react faintly for AChE and intensely for cytochrome oxidase. They contain crest synapses formed by two habenular afferents, one from each medial habenula, which contact a narrow dendritic process en passant. The lateral subnuclei react intensely for AChE and have ultrastructural features similar to the rostral subnuclei. The interstitial subnuclei lie within each fasciculus retroflexus as it enters IPN. The small dorsal lateral subnuclei are evident by light microscopy.     

Nerve growth factor receptor-immunoreactive neurons within the developing human cortex.

A monoclonal antibody recognizing the p75 receptor for nerve growth factor (NGF) was used to assess the immunohistochemical expression of NGF receptors within the developing human neo-, limbic, and paralimbic cortices as well as the hippocampal complex. Between embryonic weeks 16 and 26, a transient population of neurons located within the upper and lower subplate zones of the neo-, limbic, and paralimbic cortices expressed the receptor for NGF. In contrast, NGF receptor-immunoreactive neurons were only observed in the upper subplate zone of the entorhinal cortex at embryonic week 40 (term), a staining pattern not observed in a 5-year-old specimen. The expression of NGF receptor-immunoreactive neurons within the upper subplate zone between embryonic weeks 16 and 40 was characterized by a dense band of immunoreactive neurons and neuropil. These neurons were bipolar with basal and apically directed neurites. NGF receptor-immunoreactive neurons were also scattered throughout the lower subplate zone and underlying white matter between embryonic weeks 19 and 26. These neurons were multipolar, with less apically directed neurites. NGF receptor-immunoreactive subplate neurons displayed a topographic distribution with the heaviest concentration found within limbic and paralimbic cortices as well as association neocortex. In contrast, light to moderate NGF receptor-immunoreactivity was seen in sensory-motor cortex. Within the hippocampal complex, only a few lightly stained NGF receptor-immunoreactive neurons were seen within the fimbria, hilar region of the dentate gyrus, and subiculum. The expression of NGF receptor-immunoreactivity increased within the subplate zone of the pre- and parasubiculum culminating in intense entorhinal cortex staining. As the entorhinal cortex merged with the developing inferior temporal association cortex, there was a marked reduction in staining intensity. In contrast to those in the subplate zone, neurons within the germinal zone and cortical plate were NGF receptor immunonegative at all times examined. The presence of NGF receptors in the subplate zone suggests that neurotrophins such as NGF play an important role in the transient viability of these neurons as well as in the guidance of cortical afferent inputs into topographically organized regions of the cerebral cortex.     

The septal EEG suggests a distributed organization of the pacemaker of hippocampal theta in the rat

Individual neurons in the medial septum and diagonal band fire in phase with, and appear to act as a 'pacemaker' of, the hippocampal theta rhythm. We investigated the relationships of periodic EEG both among various parts of the septum and with dorsal hippocampal theta recorded concurrently in freely moving rats. Most septal sites showed theta rhythm concurrent with hippocampal theta during locomotion. However, periods with theta at hippocampal but not septal sites were more frequent than the reverse. Theta waves in different parts of the septum were synchronized with each other but medial septal sites showed less frequent theta than other sites. The phase delays between medial and lateral septal sites were < 10 ms, suggesting that the hippocampus does not act as a simple relay between the two. Spectral analysis revealed periods (> 5 s) of theta at hippocampal sites co-occurring with rhythms at multiple septal sites that were slower than theta. Even slower were the 'slow septal waves' (mean 2.7 Hz), which were present in the absence of locomotion and did not 'drive' the hippocampus. Our data suggest that the pacemaker of hippocampal theta may best be thought of as a set of functionally differentiated components rather than as a single homogenous unit.     

Similarities in the ultrastructural distribution of nerve growth factor receptor-like immunoreactivity in cerebellar Purkinje cells of the neonatal and colchicine-treated adult rat.

The intracellular distribution of nerve growth factor receptor immunoreactivity was examined by electron microscopy in the cerebellum of adult and postnatal day 12 rats. The very faint immunostaining in Purkinje cells of naive adult animals was greatly amplified after colchicine treatment. Neonatal cerebellum, in contrast, contained prominent immunoreactivity in both Purkinje cells and germinal cells of the external granular layer. Intracellular distribution of the nerve growth factor receptor reaction product was very similar in Purkinje cells of both neonatal and colchicine-treated adult animals. It was consistently present along the perikaryal cell membrane, in segments of the rough endoplasmic reticulum and Golgi complex. Numerous membrane-bound aggregates of immunoreactive vesicles resembling multivesicular bodies (secondary lysosomes) were scattered throughout the cell soma, although less frequently in neonatal rats. Bulbous expansions along the proximal axons of colchicine-treated Purkinje cells were filled with such immunoreactive multivesicular bodies. These cells also displayed evidence of nerve growth factor receptor internalization in the form of immunoreactive coated vesicles situated near the cell membrane. In addition to the staining in Purkinje cells, neonatal cerebellum contained high amounts of nerve growth factor receptor reaction product along the cell membrane of germinal cells in the external granular layer. Although Purkinje cells of naive adult animals possessed little or no cell membrane-related nerve growth factor receptor immunoreactivity, reaction product was sometimes seen in cisternae of the rough endoplasmic reticulum and Golgi apparatus. These findings provide electron microscopic immunocytochemical evidence of nerve growth factor receptor synthesis, internalization, and catabolism in noncholinergic neurons of the central nervous system.