L-ephedrine-induced neurodegeneration in the parietal cortex and thalamus of the rat is dependent on hyperthermia and can be altered by the process of in vivo brain microdialysis.

Multiple doses of the dietary supplement L-ephedrine can cause severe hyperthermia and modest dopamine depletions in the rat brain. Since D-amphetamine treatment can result in neurodegeneration, the potential of L-ephedrine to produce similar types of degeneration was investigated. Adult male rats, some implanted in the caudate/putamen (CPu) for microdialysis, were given four doses of 25 mg/kg L-ephedrine or 5 mg/kg D-amphetamine (2 h between doses) at an ambient temperature of 23 degrees C. L-ephedrine-induced degeneration in the forebrain was dependent on the degree of hyperthermia. Layer IV of the parietal cortex was the most sensitive to L-ephedrine treatment with peak body temperatures of at most 40.0 degrees C necessary to produce degeneration. Extensive neurodegeneration in the parietal cortex after L-ephedrine treatment was as pronounced as that previously described for D-amphetamine treatment and also occurred in the intralaminar, ventromedial and ventrolateral thalamic nuclei in rats with severe hyperthermia (peak body temperatures>41.0 degrees C). The neurodegeneration induced by L-ephedrine may have resulted in part from excitotoxic mechanisms involving the indirect pathways of the basal ganglia and related areas. No differences were observed between microdialysis and non-implanted rats with respect to degree of tyrosine hydroxylase (TH) loss in the CPu after either D-amphetamine or L-ephedrine treatment. However, neurodegeneration resulting from D-amphetamine and L-ephedrine was reduced in the microdialysis animals in the hemisphere ipsilateral to the probe, which raises concerns when using the technique of in vivo microdialysis to evaluate neurodegeneration. The results of this study, in conjunction with human clinical evaluation of ephedrine neurotoxicity, indicate that regionally specific damage may occur in the cortex of some humans exposed to ephedrine in the absence of stroke or hemorrhage.

Muscarinic m1 and m2 receptor proteins in local circuit and projection neurons of the primate striatum: anatomical evidence for cholinergic modulation of glutamatergic prefronto-striatal pathways.

The cellular and subcellular localization of muscarinic receptor proteins m1 and m2 was examined in the neostriatum of macaque monkeys by using light and electron microscopic immunocytochemical techniques. Double-labeling immunocytochemistry revealed m1 receptors in calbindin-D28k--positive medium spiny projection neurons. Muscarinic m1 labeling was dramatically more intense in the striatal matrix compartment in juvenile monkeys but more intense in striosomes in the adult caudate, suggesting that m1 expression undergoes a developmental age-dependent change. Ultrastructurally, m1 receptors were predominantly localized in asymmetric synapse-forming spines, indicating that these spines receive extrastriatal excitatory afferents. The association of m1-positive spines with lesion-induced degenerating prefronto-striatal axon terminals demonstrated that these afferents originate in part from the prefrontal cortex. The synaptic localization of m1 in these spines indicates a role of m1 in the modulation of excitatory neurotransmission. To a lesser extent, m1 was present in symmetric synapses, where it may also modulate inhibitory neurotransmission originating from local striatal neurons or the substantia nigra. Conversely, m2/choline acetyltransferase (ChAT) double labeling revealed that m2-positive neurons corresponded to large aspiny cholinergic interneurons and ultrastructurally, that the majority of m2 labeled axons formed symmetric synapses. The remarkable segregation of the m1 and m2 receptor proteins to projection and local circuit neurons suggests a functional segregation of m1 and m2 mediated cholinergic actions in the striatum: m1 receptors modulate extrinsic glutamatergic and monoaminergic afferents and intrinsic GABAergic afferents onto projection neurons, whereas m2 receptors regulate acetylcholine release from axons of cholinergic interneurons.

Estrogen receptor beta immunoreactivity in differentiating cells of the developing rat cerebellum.

Estrogen receptors (ER) play a significant role in the development of some regions of the mammalian brain. Recently, ER-beta (ERbeta) mRNA and protein were shown to be expressed in the rat cerebellum. In the present study, the ontogeny of ERbeta protein expression was examined in the rat cerebellum during postnatal development. Western blot analysis indicated that a single ERbeta-like immunoreactive species of approximately 55 kDa was present in protein lysates prepared from the cerebella of female and male Sprague-Dawley rat pups. Immunocytochemical analysis of cerebellar sections from the midline vermis revealed that during development, the expression of ERbeta varied with age and cell-type, but not sex. In the developing cerebellum, highest levels of ERbeta-immunoreactivity (IR) were detected in neurons during neurite growth, and in some glia during migration. Throughout the first postnatal week, ERbeta-IR was localized to differentiating granule cells in the external germinal layer and to migrating glia. Differentiating granule cells expressed detectable levels of ERbeta throughout development. In Purkinje cells, ERbeta-IR was first detected on postnatal day 6 (P6), with peak intensities of immunostaining coinciding with the initiation of axonal and dendritic growth that occurs between P7 and P8. Expression of ERbeta-IR remained high during maturation of Purkinje cell dendrites, and then decreased to a lower level maintained in the adult. From the third postnatal week, ERbeta-IR was also detected in the later developing Golgi, stellate, and basket neurons. These results suggest that ERbeta may play a role in growth-related mechanisms during differentiation of cerebellar neurons and glia.

Segregation of serotonin 5-HT2A and 5-HT3 receptors in inhibitory circuits of the primate cerebral cortex.

An emerging concept of cortical network organization is that distinct segments of the pyramidal neuron tree are controlled by functionally diverse inhibitory microcircuits. We compared the expression of two serotonin receptor subtypes, the G-protein-coupled 5-hydroxytryptamine2A receptors and the ion-channel gating 5-HT3 receptors, in cortical neuron types, which control these microcircuits. Here we show, using light and electron microscopic immunocytochemical techniques, that 5-HT2A receptors are segregated from 5-HT3 receptors in the macaque cerebral cortex. 5-HT2A receptor immunolabel was found in pyramidal cells and also in GABAergic interneurons known to specialize in the perisomatic inhibition of pyramidal cells: large and medium-size parvalbumin- and calbindin-containing interneurons. In contrast, 5-HT3 label was only present in small GABA-, substance P receptor-, and calbindin-containing neurons and in medium-size calretinin-containing neurons: interneurons known to preferentially target the dendrites of pyramidal cells. This cellular segregation indicates a serotonin-receptor-specific segmentation of the GABAergic inhibitory actions along the pyramidal neuron tree.

5-Hydroxytryptamine2A serotonin receptors in the primate cerebral cortex: possible site of action of hallucinogenic and antipsychotic drugs in pyramidal cell apical dendrites.

To identify the cortical sites where 5-hydroxytryptamine2A (5-HT2A) serotonin receptors respond to the action of hallucinogens and atypical antipsychotic drugs, we have examined the cellular and subcellular distribution of these receptors in the cerebral cortex of macaque monkeys (with a focus on prefrontal areas) by using light and electron microscopic immunocytochemical techniques. 5-HT2A receptor immunoreactivity was detected in all cortical layers, among which layers II and III and layers V and VI were intensely stained, and layer IV was weakly labeled. The majority of the receptor-labeled cells were pyramidal neurons and the most intense immunolabeling was consistently confined to their parallelly aligned proximal apical dendrites that formed two intensely stained bands above and below layer IV. In double-label experiments, 5-HT2A label was found in calbindin D28k-positive, nonphosphorylated-neurofilament-positive, and immuno-negative pyramidal cells, suggesting that probably all pyramidal cells express 5-HT2A receptors. 5-HT2A label was also found in large- and medium-size interneurons, some of which were immuno-positive for calbindin. 5-HT2A receptor label was also associated with axon terminals. These findings reconcile the data on the receptor's cortical physiology and localization by (i) establishing that 5-HT2A receptors are located postsynaptically and presynaptically, (ii) demonstrating that pyramidal neurons constitute the major 5-HT2A-receptor-expressing cells in the cortex, and (iii) supporting the view that the apical dendritic field proximal to the pyramidal cell soma is the "hot spot" for 5-HT2A-receptor-mediated physiological actions relevant to normal and "psychotic" functional states of the cerebral cortex.

Quantitative three-dimensional analysis of the catecholaminergic innervation of identified neurons in the macaque prefrontal cortex.

The present study provides a complete quantitative three-dimensional analysis of neurons in primate prefrontal cortex targeted by catecholaminergic axons. Individual pyramidal and nonpyramidal cells in fixed slices were filled with Lucifer yellow (LY) and recovered with anti-LY antibody combined with anti-tyrosine hydroxylase (TH) antisera to reveal catecholaminergic axons. The total number of TH contacts and TH apposition density (THAD) was obtained for pyramidal and nonpyramidal cells in different layers. Four TH contacts (two on spines and two on shafts) were selected for correlated electron microscopic examination and serially sectioned; all four were confirmed as membrane appositions. Quantitative analysis revealed 90 TH contacts per pyramidal neuron in layer III, with a density of 0.8 per 100 microm of dendritic length (i.e., averaging one contact per basal dendrite). Remarkably, pyramids of layers III, V, and VI had the same THAD values, with a highly regular distribution of TH terminals on their spiny dendritic trees. In contrast, TH contacts on nonpyramidal neurons in layer III were half as dense and, moreover, were distributed irregularly and showed large variation from cell to cell. Neurons in layers II and superficial III had the highest THAD, as compared with deeper layers (1.4 vs 0.7 per 100 micron of dendritic length for pyramids; 0.53 vs 0.4 for interneurons). The highly organized TH innervation of pyramidal neurons, with at least one contact on virtually every dendrite, indicates that catecholaminergic, presumably dopaminergic, terminals are placed strategically along the entire dendritic tree to modulate most, if not all, of the excitatory input of a neuron. At the same time, the sparsity of contacts per dendrite may explain cortical vulnerability in diseases involving dopamine.

Dual role of substance P/GABA axons in cortical neurotransmission: synaptic triads on pyramidal cell spines and basket-like innervation of layer II-III calbindin interneurons in primate prefrontal cortex.

In spite of accumulating evidence on the potent neuromodulatory, neuroprotective, trophic and memory-enhancing effects of the neuropeptide substance P (SP) in the cerebral cortex, the excitatory or inhibitory nature of the cortical SP innervation remains unclear and the postsynaptic targets of SP fibers are not defined. To obtain further insight into these issues, we have examined SP-containing axons and their postsynaptic targets in the prefrontal cortex of adult monkeys with single- and double label immunocytochemistry combined with light and correlated electron microscopy. SP fibers in the primate prefrontal cortex, unlike those in the rat cortex, preferentially innervate cortical layers I, II and upper layer III. Our results demonstrate for the first time that all SP-immunoreactive boutons in all cortical layers contain GABA. Of the entire sample of SP boutons, 53% synapse on dendritic shafts, 39% on dendritic spines and 8% on cell bodies. Another new finding is that synapse-forming SP boutons, in addition to their known innervation of pyramidal cells, form pericellular baskets around interneurons in layers II and upper III, a subpopulation of which contains calbindin D28k. Finally, the study also revealed that SP boutons frequently participate in 'synaptic triads' with spines which receive another (asymmetric, putatively excitatory amino acid-utilizing) synapse. Our findings indicate that SP/GABA axons in the primate prefrontal cortex modulate excitatory amino acid-mediated neurotransmission and control feed-forward disinhibitory GABAergic circuits in supragranular cortical layers.

Aromatase in axonal processes of early postnatal hypothalamic and limbic areas including the cingulate cortex.

It has been shown that sexual dimorphic morphology of certain hypothalamic and limbic areas underlie gender-specific sexual behavior and neuroendocrine mechanisms. The key role played by locally formed estrogen in these developmental events has been revealed during a critical perinatal period. In this study, we aimed to document the presence of estrogen-synthetase (aromatase)-immunoreactive elements in the involved limbic system and hypothalamus of the developing rat brain. On postnatal day 5, animals of both sexes were perfusion-fixed, and sections from the forebrain and hypothalamus were immunolabelled for aromatase using an antiserum that was generated against a 20 amino acid sequence of placental aromatase. Aromatase-immunoreactivity was present in neuronal perikarya and axonal processes in the following limbic structures: the central and medial nuclei of the amygdala, stria terminalis, bed nucleus of the stria terminalis (BNST), lateral septum, medial septum, diagonal band of Broca, lateral habenula and all areas of the limbic (cingulate) cortex. In the hypothalamus, the most robust labelling was observed in the medial preoptic area, periventricular regions, ventromedial and arcuate nuclei. The most striking feature of the immunostaining with this antiserum was its intracellular distribution. In contrast to the heavy perikaryal labelling that can be observed with most of the currently available aromatase antisera, in the present experiments, immunoperoxidase was predominantly localized to axons and axon terminals. All the regions with fiber staining corresponded to the projection fields of neuron populations that have previously been found to express perikaryal aromatase. Our results confirm the presence of aromatase-immunoreactivity in developing limbic and hypothalamic areas. The massive expression of aromatase in axonal processes raises the possibility that estrogen formed locally by aromatase may not only regulate the growth, pathfinding and target recognition of its host neuronal processes, but may also exert paracrine actions on structures in close proximity, including the target cells.

Morphogenetic plasticity of neuronal elements in cerebellar glomeruli during deafferentation-induced synaptic reorganization.

Reorganization of the cerebellar glomerulus, the main synaptic complex within the granule cell layer, was investigated using quantitative morphological techniques. All afferents to the cerebellar cortex, including mossy-fibers, were surgically destroyed by undercutting the cerebellar vermis. Fifteen days after the operation, which resulted in the removal of the main excitatory afferent to the glomerulus, a significant reorganization of the whole synaptic complex was observed, whereas the structural integrity of the glomerulus was remarkably well preserved. This was indicated by the observation that the number of granule cell dendrites (approximately 50 per glomerulus), as well as the number of dendritic digits (approximately 210 per glomerulus) bearing most of the approximately 230 synaptic junctions per glomerulus, did not change significantly after mossy-fiber degeneration. The total number of synapses in the reorganized glomerulus did not change either, despite the disappearance of two-thirds of (excitatory) synaptic junctions caused by mossy-fiber degeneration. In the reorganized glomeruli, however, the inhibitory, GABA-containing Golgi axonal varicosities became the dominant synaptic type-about four-fifths (approximately 200) of all synapses within the glomerulus-whereas the dendritic synapses between the granule cells represented only one-fifth of all synaptic junctions. The quantitative data of the reorganized cerebellar glomerulus demonstrate both a remarkable constancy and a plasticity of the excitatory granule cells and inhibitory Golgi neurons building up this synaptic complex. Constancy (the preservation of certain specific structural features) is represented by an eventually unchanged number of dendrites and synaptic junctions within the deafferented glomerulus. Such constancy was made possible, however, by the morphogenetic plasticity of both nerve-cell types to produce new, dendro-dendritic and axo-dendritic synapses to compensate for the loss of mossy-fiber synapses.

Presynaptic and postsynaptic subcellular localization of substance P receptor immunoreactivity in the neostriatum of the rat and rhesus monkey (Macaca mulatta).

The substance P receptor (SPR) gene is expressed at high levels in basal ganglia, but the paucity of information about localization of the encoded receptor protein has limited our understanding of this peptide's involvement in cellular and subcellular mechanisms in this region. Morphological evidence in the rodent striatum indicates that SPRs are expressed in postsynaptic neuronal elements, while pharmacological studies suggest the existence of presynaptic SPRs in this structure. We have examined the issue of subcellular distribution of this receptor protein in rat and primate neostriatal tissue, employing an antiserum raised against SPR. Electron microscopic analysis revealed that SPR immunoreactivity is present in presynaptic and postsynaptic neuronal elements in both species. In agreement with earlier studies, SPR immunoreactivity was found predominantly in perikarya and dendrites of a small subset of striatal neurons, the large and medium-sized aspiny interneurons. In addition, a small but significant proportion of the immunoreaction product was localized in presynaptic profiles, both in axons and axon terminals. The majority of SPR immunoreactive boutons formed asymmetric synapses with dendrites and dendritic spines. The association of SPRs with asymmetric synapses provides a morphological substrate for peptidergic modulation of excitatory neurotransmission of extrastriatal origin. A minor proportion of immunolabeled axons established symmetric synaptic junctions with unlabeled dendrites. The presence of SPRs in these synapses suggests a presynaptic peptidergic modulation of intrinsic striatal transmitter systems. The observations in this study also indicate that SPR mediates a complex combination of postsynaptic and presynaptic effects on acetylcholine release in the mammalian striatum.

Distribution and neurochemical character of substance P receptor (SPR)-immunoreactive striatal neurons of the macaque monkey: accumulation of SP fibers and SPR neurons and dendrites in "striocapsules" encircling striosomes.

The striatal distribution of the substance P receptor (SPR) protein was examined in relation to its ligand, the neuro-peptide SP, as well as to the neurochemical and compartmental composition of the neostriatum in rhesus monkeys (Macaca mulatta) in immunohistochemical experiments. About 2% of striatal neurons, displaying varicose, virtually spine-free dendrites characteristic of large and medium-sized aspiny interneurons, expressed SPR immunoreactivity. SPR/choline acetyltransferase, SPR/somatostatin, SPR/GABA, SPR/calbindin D28k, and SPR/parvalbumin double immunolabeling experiments demonstrated that SPR-positive cells are either cholinergic or somatostatinergic. Comparison of SP and SPR immunoreactivities in double-labeled and adjacent single-labeled sections revealed compartment-specific match and mismatch between the densities of the peptide and receptor. A matching high density of SP fibers and SPR cells and dendrites was only observed in the rim of the striosome compartments. To our knowledge, this is the first evidence for an anatomical border comprised of dendritic processes that separate striatal compartments. We have termed these zones "striocapsules," because they encircle and encapsulate striosomal cell islands. In the striatal matrix, an abundance of SPR-labeled profiles was complemented with light SP staining. By contrast, in the core of the striosomes, SPR labeling was sparse and SP staining intense. SP-positive axon-like puncta frequently contacted SPR-positive dendrites in all striatal compartments. The SP receptor/ligand match indicates a sharp increase in the efficacy of SP action in the striocapsules, and suggests that the influence of SP might be heightened in this striatal subcompartment.

Aromatase immunoreactivity in axon terminals of the vertebrate brain. An immunocytochemical study on quail, rat, monkey and human tissues.

Intraneuronal production of estradiol from testosterone has been shown to play a pivotal role in gender-specific brain development of most vertebrates, and to participate in numerous functions of the adult central nervous system. Previous biochemical and morphological approaches demonstrated that estrogen synthetase (aromatase) is present in specific limbic and hypothalamic structures. On the other hand, less attention has been paid to revealing its subcellular distribution. The possibility of aromatase presence in axonal processes has been indicated by recent biochemical and morphological observations suggesting new insights for the role of aromatase in neural functions. The objective of the present study was to provide morphological evidence for the subcellular location of aromatase in neurons of different vertebrate species including Japanese quail, rat, monkey, and human. Immunocytochemistry using a purified polyclonal antiserum against human placental aromatase localized immunoreactivity to hypothalamic and limbic cell groups in all of these species. Light and electron microscopic examination of vibratome sections revealed the presence of aromatase immunoreactivity throughout the neuronal perikarya, including dendrites and axonal processes. In each species there were numerous boutons which contained labeled small clear synaptic vesicles. Many of these axon terminals formed synapses with immuno-negative and immuno-positive dendrites and perikarya. This study furnishes the first immunolocalization of aromatase in the brains of two primate species, humans and monkeys. The provision of further evidence for estrogen synthesis in axons and axon terminals may help resolve apparent differences between the measurement of aromatase activity and the lack of aromatase-immunopositive cell bodies in previous studies. The present findings may be coupled with recent evidence regarding the molecular biology and the diversity of functional properties of P450 aromatase to indicate previously unexpected effects of brain aromatase at the synaptic level.

Extrinsic and intrinsic substance P innervation of the rat lateral septal area calbindin cells.

The electrophysiological observations that substance P administration to the lateral septal area elicits both excitatory and inhibitory responses, together with earlier reports on the multiple sources of substance P innervation of the septum, implies that these axons with distinct origins have different functions. This prompted us to examine the origin and neurochemical character of substance P afferents to the lateral septal area. Chronic surgical isolation of the septum from its ventral afferents and retrograde tracer experiments using wheat germ agglutinin-conjugated horseradish peroxidase, both followed by an immunostaining for substance P, were employed to elucidate the origin of these axon terminals. In order to assess the possible co-existence of substance P with other neurotransmitter substances in the parent cells of the septopetal projections, co-localization studies for substance P and choline acetyltransferase, as well as substance P and GABA, were performed. The comparative distribution of substance P fibers and septal calbindin-containing neurons was also investigated using correlated light and electron microscopic double immunostaining. The results are summarized as follows: (i) the substance P innervation of the lateral septal area derives from several hypothalamic nuclei (including the lateral and lateroanterior hypothalamic area, tuber cinereum and ventromedial hypothalamic nucleus) and tegmental nuclei (the majority of fibers from the laterodorsal and a few from the pedunculopontine tegmental nucleus), as well as intrinsic septal cells; (ii) the septopetal substance P fibers of tegmental origin are cholinergic; intraseptal substance P neurons located in the dorsolateral part of the lateral septum also contain GABA, while substance P neurons seen on the border between the medial and lateral septal area and septopetal hypothalamic substance P cells do not contain GABA or acetylcholine; (iii) substance P fibers from pericellular baskets around calbindin-containing lateral septal neurons with a high degree of selectivity; (iv) approximately 90% of the entire calbindin cell population are postsynaptic targets of substance P axons; (v) their terminals contact the soma and the dendrites of these cells, among them the somatospiny neurons; and (vi) the extrinsic substance P boutons establish asymmetric, while the intrinsic substance P axon terminals form symmetric membrane specializations. Because neurons in the lateral septal area receive hippocampal input and project massively to hypothalamic areas, the different types of substance P input on these neurons can modify the information flow arriving from the hippocampus to diencephalic brain structures at the level of the lateral septal area.

Aromatase- (estrogen synthetase) immunoreactive neurons in the rat septal area. A light and electron microscopic study.

The aromatase enzyme (estrogen synthetase) catalyzes the conversion of testosterone to estrogen in peripheral and central nervous tissue. Light and electron microscopic immunocytochemistry was used to study the localization of this enzyme in the septal area of adult male and female albino rats. Aromatase-immunoreactivity was found restricted to neuronal somata and dendritic arbors, and no sex differences were detected in its distribution or intensity. Most aromatase-immunoreactive neurons formed two oblique bands in the lateral and the medial zones of the lateral septum; in addition, labeled cells were present in the septohippocampal nucleus and the laterodorsal portion of the bed nucleus of the stria terminalis. Electron microscopy revealed that the majority of aromatase-positive neurons in the lateral septum exhibit somatic spines, a characteristic marker of a neuron population that is known to contribute to local and extraseptal projections. The presence of aromatase in lateral septal somatospiny neurons suggests that estrogen formed by these neurons may be critically involved in the septal control of steroid-dependent behaviors.

Aromatase immunoreactivity in the rat brain: gonadectomy-sensitive hypothalamic neurons and an unresponsive "limbic ring" of the lateral septum-bed nucleus-amygdala complex.

The aromatase (estrogen synthetase) enzyme catalyzes the conversion of androgens to estrogens in peripheral tissues, as well as in the brain. Our study aimed at comparing the brain distribution of aromatase-immunoreactive neurons in male and female, normal and gonadectomized rats. Light microscopic immunostaining was employed using a purified polyclonal antiserum raised against human placental aromatase. Two anatomically separate aromatase-immunoreactive neuronal systems were detected in the rat brain: A "limbic telencephalic" aromatase system was composed by a large population of labeled neurons in the lateral septal area, and by a continuous "ring" of neurons of the laterodorsal division of the bed nucleus of stria terminalis, central amygdaloid nucleus, stria terminalis, and the substantia inominata-ventral pallidum-fundus striati region. The other, "hypothalamic" aromatase system consisted of neurons scattered in a dorsolateral hypothalamic area including the paraventricular, lateral and dorsomedial hypothalamic nuclei, the subincertal nucleus as well as the zona incerta. In addition, a few axon-like processes (unresponsive to gonadectomy) were present in the preoptic-anterior hypothalamic complex, the ventral striatum, and midline thalamic regions. No sexual dimorphism was observed in the distribution or intensity of aromatase-immunostaining. However, 3 days, 2, 3, 8, 16, or 32 weeks after gonadectomy, aromatase-immunoreactive neurons disappeared from the hypothalamus, whereas they were still present in the limbic areas of both sexes. The results indicate the existence of two distinct estrogen-producing neuron systems in the rat brain: (1) a "limbic ring" of aromatase-labeled neurons of the lateral septum-bed nucleus-amygdala complex unresponsive to gonadectomy; and (2) a sex hormone-sensitive "hypothalamic" aromatase neuron system.

Presence of somatostatin or neurotensin in lateral septal dopaminergic axon terminals of distinct hypothalamic and midbrain origins: convergence on the somatospiny neurons.

In the lateral septal area (LSA), both inhibitory and excitatory dopamine (DA) actions, as well as hypothalamic and midbrain DA efferents, have been described. Some neurons of the hypothalamic and midbrain DA systems contain somatostatin (SOM) or neurotensin (NT), and, in the LSA, the distribution of fibers containing these peptides overlaps with DA fibers. These data prompted us to test for the presence of SOM and NT in LSA dopaminergic axon terminals of hypothalamic and midbrain origins. To verify the origins of SOM and NT innervation of the LSA, the retrograde tracer horseradish peroxidase conjugated with wheat germ agglutinin (HRP-WGA) was injected into the LSA, and alternate brain sections were immunostained for SOM, NT, or tyrosine hydroxylase (TH) in group 1 rats. Numerous retrogradely labeled neurons were found immunopositive for SOM in the periventricular and basolateral hypothalamus, many HRP-WGA labeled cells contained NT immunoreactivity in the ventral tegmental area, and TH-immunoreactive retrogradely labeled neurons were observed in both brain areas. In a new approach, the presence of these peptides in dopaminergic boutons was assessed by combining peptide immunocytochemistry with acute 6-hydroxydopamine (6-OHDA) induced lesioning of DA cell groups. These groups of rats were treated with desipramine to protect the noradrenergic fibers, and 45 min later 1 microgram 6-OHDA (in 0.5 microliter saline) was unilaterally injected into the periventricular hypothalamus (group 2) or the ventral tegmental area (group 3). After 48 h the rats were killed and alternate septal sections of both groups were immunostained for TH, SOM, or NT. On the operated side of the LSA in both groups, electron microscopy revealed numerous axon terminals that were immunopositive for TH and contained autophagous cytolysosomes, an early sign of catecholamine fiber degeneration induced by 6-OHDA. In group 2, phagosome-containing boutons were found immunopositive for SOM, but not for NT; vice versa, in group 3, only NT-positive degenerated boutons were detected. SOM- and NT-positive degenerated axon terminals in both groups formed synaptic contacts with LSA neurons, in particular with somatospiny cells. On the contralateral side of the LSA, all of the axon terminals were intact. It has been shown that SOM exerts an inhibitory action, whereas NT has an excitatory effect on limbic area neurons. Thus, the results implicate that the differential peptide content of dopamine fibers marks their functional differences. It appears that LSA neurons receive double innervation from an inhibitory "somatostatinergic" DA system of the hypothalamus, and from an excitatory "neurotensinergic" DA system of the midbrain.

Synaptology and origin of somatostatin fibers in the rat lateral septal area: convergent somatostatinergic and hippocampal inputs of somatospiny neurons.

This study deals with the synaptology, morphologically identified postsynaptic targets, and origin of somatostatin (SOM) fibers in the rat lateral septal area (LSA) with special reference to those forming pericellular baskets. Septal vibratome sections were immunostained for SOM-14 in 3 experimental groups: control animals, rats subjected to a chronic transection of the ascending afferents to the septum, and animals with acute fimbria-fornix lesion. Light microscopy revealed that the SOM-immunoreactive fibers form pericellular baskets predominantly in the intermediate and ventral parts of the caudal half of the LSA. Electron microscopic analysis showed that the somatospiny neurons are postsynaptic targets of these pericellular baskets. Eight days after a unilateral cut placed at the ventral border of the septum, virtually all SOM-immunoreactive axon terminals disappeared from the ipsilateral intermediate and ventral LSA, and they were substantially reduced in the dorsal LSA. However, in these rats SOM-positive neurons could be observed in the LSA on the lesioned, but not on the contralateral side. Furthermore, on the lesion side of the anterior periventricular hypothalamus an increase was detected both in the number and the intensity of immunostaining of SOM-positive neurons. Thirty-six h following a unilateral transection of the fimbria-fornix, the SOM-immunoreactive axon terminals in the LSA remained intact; only immunonegative degenerated hippocamposeptal boutons were detected forming synaptic contacts with somatospiny neurons. Axosomatic synapses of SOM-positive boutons regularly appeared at the neck of somatic spines which were postsynaptic to degenerated hippocamposeptal fibers. The results indicate that the septal SOM fibers are of multiple origin. Those forming pericellular baskets in the LSA originate in ventral extraseptal, probably periventricular hypothalamic areas. SOM fibers scattered in the dorsal LSA are most likely processes of local SOM neurons. The accumulation of immunoreactive SOM in some cells of the undercut septum is a sign of axonal lesion, indicating that these neurons project outside the septum. The SOM innervation of somatospiny neurons which also receive hippocampal input and have been reported to contain gamma-aminobutyric acid (GABA) may be a morphological substrate of the SOM-related disinhibition in the LSA.

Convergent vasopressinergic and hippocampal input onto somatospiny neurons of the rat lateral septal area.

Electron microscopic immunocytochemistry, was combined with acute anterograde axon degeneration, following transection of the fimbria-fornix, to describe the innervation of somatospiny neurons by vasopressin-immunoreactive and degenerated hippocamposeptal axon terminals in the rat lateral septal area. Vasopressin-immunopositive boutons characterized by symmetric synaptic membrane specializations, and the degenerated hippocamposeptal axon terminals which form asymmetric synaptic contacts, frequently terminate on the same dendritic and somatic profiles, and particularly on the somata of somatospiny neurons. Although hippocamposeptal fibers predominantly form axospinous synapses in the lateral septal area, they terminate mainly on the dendritic shafts and soma of the vasopressin-receptive neurons. Of 720 vasopressin-immunoreactive terminals in the mediolateral part of the lateral septal area, 80% form synaptic contacts with dendritic shafts; 50% on small (distal) dendritic profiles and 30% on large (proximal) dendrites. Synaptic contacts between vasopressin-immunoreactive terminals and dendritic spines were not observed. The remaining 20% of immunoreactive boutons formed axosomatic synaptic contacts with a total of 58 neurons; 31% of these neurons exhibited somatic spines in the plane of the section analysed. Previous studies have demonstrated that in the lateral septal area vasopressin modulates the action of the excitatory amino acid-containing hypocamposeptal fibers, and also plays a role in the maintenance of long term potentiation evoked by fimbria-fornix stimulation. The convergent vasopressinergic and hippocampal input onto the same somatospiny neurons of the lateral septal area suggests that these neurons are targets of these physiological actions.

Catecholaminergic, GABAergic, and hippocamposeptal innervation of GABAergic "somatospiny" neurons in the rat lateral septal area.

This study deals with the neurochemical characterization of the rat lateral septal area (LSA) somatospiny neurons and their innervation by hippocamposeptal, catecholaminergic, and GABAergic fibers. Electron microscopic single and double immunostaining methods were used to label catecholaminergic fibers and GABAergic cells and boutons. Axon terminals originating in the hippocampus were labeled by acute anterograde axon degeneration induced by fimbria-fornix transection 36 hours before sacrifice. Three types of experiments were performed. The convergent catecholaminergic and hippocamposeptal innervation of LSA somatospiny neurons was studied by combining immunostaining for tyrosine hydroxylase (TH) with fimbria-fornix transection. GABAergic neurons and their hippocamposeptal afferents were identified and characterized in colchicine pretreated animals immunostained for glutamic acid decarboxylase (GAD) combined with fimbria-fornix transection. The third experiment aimed at simultaneously visualizing the relationships between catecholaminergic boutons, hippocamposeptal excitatory amino acid containing axon terminals and GABAergic profiles by double immunostaining for TH (the PAP technique) and GAD (the immunogold method) combined with fimbria-fornix transection. The results are summarized as follows: 1) The same LSA somatospiny neurons receive synaptic inputs from the hippocampus and TH immunoreactive fibers which form pericellular baskets around these cells. 2) LSA somatospiny neurons are GABAergic and are postsynaptic targets of GABAergic boutons with unknown origin and hippocamposeptal axon terminals. 3) The double immunostaining experiment, finally, provided direct evidence that the same GABAergic somatospiny neurons are postsynaptic targets of both catecholaminergic and hippocamposeptal afferents. The synaptic interconnections described in this study provide anatomical basis for a better understanding of the action of catecholamines, excitatory amino acids, and GABA on the activity of LSA neurons.