Group II metabotropic glutamate receptors in anxiety circuitry: correspondence of physiological response and subcellular distribution.

Activation of group II metabotropic glutamate receptors (mGluR2/3) in the amygdala plays a critical role in the regulation of fear and anxiety states. Previous studies using nonselective agonists have suggested this action can result from activation of either pre- or postsynaptic mGluR2/3. Here, we have used a combination of whole-cell patch clamp recording with highly selective agonists (LY354740 and LY379268) and immunoelectron microscopy to examine structure-function relationships for mGluR2/3 in the basolateral amygdala (BLA) and bed nucleus of the stria terminalis (BNST). Stimulation of mGluR2/3 evoked a direct, TTX-insensitive membrane hyperpolarization in all BLA projection neurons tested, but only about half of BNST neurons. The membrane hyperpolarization was mediated by activation of an outward potassium current or blockade of a tonically active inward I(h) current in different groups of BLA neurons. In both regions, mGluR2/3 caused a long-lasting reduction of glutamate release from presynaptic afferent terminals even at concentrations that failed to elicit a direct postsynaptic response. The localization of mGluR2/3 differed regionally, with postsynaptic labeling significantly more common in BLA than BNST, corresponding to the strength of postsynaptic responses recorded there. Our results demonstrate a complex role for mGluR2/3 receptors in modulating anxiety circuitry, including direct inhibition and reduction of excitatory drive. The combination of direct inhibition of projection neurons within the BLA and suppression of excitatory neurotransmission in the BNST may be responsible for the anxiolytic actions of group II mGluR agonists.

A disynaptic pathway from the central amygdaloid nucleus to the paraventricular hypothalamic nucleus via the parastrial nucleus in the rat.

The organization of projections from the central amygdaloid nucleus (CeA) to the paraventricuilar hypothalamic nucleus (PVH) has been studied in order to understand the anatomical substrates of amygdaloid modulation of endocrine and autonomic functions, and a hypothesis that the bed nucleus of the stria terminalis (BST) may act as a relay site between the CeA and PVH has been proposed. Using anterograde and retrograde tract-tracing methods, in the rat, we first indicated that neurons in the parastrial nucleus (PS), where projection fibers from the central amygdaloid nucleus (CeA) terminated, sent their axons to the paraventricular hypothalamic nucleus (PVH). We further demonstrated that the CeA terminals formed symmetrical synaptic contacts with somata and dendrites of the PVH-projecting PS neurons, and that the PS received CeA fibers predominantly from the lateral part and sent large numbers of projection fibers to almost all the subdivisions of the PVH. Using anterograde tracing combined with the postembedding immunogold method, we finally revealed that nearly all the CeA terminals in the PS were immunoreactive for gamma-aminobutyric acid. The present data suggest that output signals from the CeA are transmitted disynaptically to the PVH neurons via the PS neurons and modulate PVH neuron activity by way of disinhibition.

Decrease in neuronal spine density in the postpartum period in the amygdala and bed nucleus of the stria terminalis in rat.

In pregnancy and the postpartum period, many women have emotional instability and some suffer from depression. The ovarian steroid hormone milieu is markedly changed during these periods, and this hormonal change may be an important cause of peripartum emotional instability. The amygdala is a central region of emotion, and the bed nucleus of the stria terminalis (BNST), which is considered to be the extended amygdala, is also involved in the emotional response. The amygdala and BNST are well characterized as target brain regions for ovarian steroid hormones, and this suggests that the functional response of neurons in these regions to hormonal fluctuation is affected in the peripartum period. In this study, we investigated the neuronal morphology in the central (CeA) and basolateral (BLA) nucleus of the amygdala and BNST on gestational days 15 (G15) (mid-gestation) and 20 (G20) (late gestation) and 4days after delivery (P4) (early postpartum) in rat. Golgi staining showed that the dendritic spine density, and particularly the number of mature mushroom-type spines, in the CeA, BLA and BNST was significantly decreased at P4, compared with G15 and G20 and with virgin females in the estrous phase in the normal estrous cycle (Est). Interestingly, the presence of pups after delivery influenced the spine density in the BNST. The density was significantly decreased with pup presence compared with pup absence at P4, and compared with G15, G20 and Est. These results provide fundamental insights into the neuronal basis underlying emotional instability during pregnancy and postpartum.

Neural interaction between galanin-like peptide (GALP)- and luteinizing hormone-releasing hormone (LHRH)-containing neurons.

Galanin-like peptide (GALP), commonly known as an appetite-regulating peptide, has been shown to increase plasma luteinizing hormone (LH) through luteinizing hormone-releasing hormone (LHRH). This led us to investigate, using both light and electron microscopy, whether GALP-containing neurons in the rat brain make direct inputs to LHRH-containing neurons. As LHRH-containing neurons are very difficult to demonstrate immunohistochemically with LHRH antiserum without colchicine treatment, we used a transgenic rat in which LHRH tagged with enhanced green fluorescence protein facilitated the precise detection of LHRH-producing neuronal cell bodies and processes. This is the first study to report on synaptic inputs to LHRH-containing neurons at the ultrastructural level using this transgenic model. We also used immunohistochemistry to investigate the neuronal interaction between GALP- and LHRH-containing neurons. The experiments revealed that GALP-containing nerve terminals lie in close apposition with LHRH-containing cell bodies and processes in the medial preoptic area and the bed nucleus of the stria terminalis. At the ultrastructural level, the GALP-positive nerve terminals were found to make axo-somatic and axo-dendritic synaptic contacts with the EGFP-positive neurons in these areas. These results strongly suggest that GALP-containing neurons provide direct input to LHRH-containing neurons and that GALP plays a crucial role in the regulation of LH secretion via LHRH.

High-frequency stimulation induces ethanol-sensitive long-term potentiation at glutamatergic synapses in the dorsolateral bed nucleus of the stria terminalis.

Anatomical and functional data support a critical role for the bed nucleus of the stria terminalis (BNST) in the interaction between stress and alcohol/substance abuse. We report here that neurons of the dorsal anterolateral BNST respond to glutamatergic synaptic input in a synchronized way, such that an interpretable extracellular synaptic field potential can be readily measured. High-frequency stimulation of these glutamatergic inputs evoked NMDA receptor (NMDAR)-dependent long-term potentiation (LTP). We found that an early portion of this LTP is reduced by acute exposure to ethanol in a GABA(A) receptor-dependent manner. This effect of ethanol is accompanied by a significant and reversible dose-dependent attenuation of isolated NMDAR signaling and is mimicked by incomplete NMDAR blockade.

Branching patterns of parabrachial neurons projecting to the central extended amgydala: single axonal reconstructions.

Electrophysiological evidence suggests that the spinoparabrachioamygdaloid pathway carries nociceptive information that may be important for the elaboration of physiological and emotional responses to noxious events. The pontine parabrachial nucleus (pPB) sends a massive projection to the central nucleus of the amygdala (CeA) and lateral bed nucleus of the stria terminalis (BSTL), both regions belonging to a broader macrostructure, the central extended amygdala (EAc). The aim of this study was to examine whether different EAc components are targeted by a same pPB neuron, by reconstructing single axonal branching patterns after anterograde labelling. Small deposits of biotinylated dextran amine in the region of the external lateral pPB result in dense and specific labelling in the whole EAc. Reconstructed axons innervate either the lateral or the capsular part of the CeA with perisomatic or bushy terminals, respectively. A subset of axons enters the stria terminalis rostrally to follow its trajectory caudally toward the CeA. Individual axons targeting the CeA usually send collaterals to other EAc components, especially those projecting to the lateral CeA, which often coinnervate the BSTL. By contrast, only few branches were found outside the EAc. These results suggest that the noxious information travelling from the pPB to the CeA may also be transmitted to other EAc components. This pPB-EAc pathway, which appears distinct from the parabrachiohypothalamic and parabrachiothalamic projections, would be the anatomical basis through which the EAc elaborates the autonomic, endocrine, and emotional components of pain.

Adolescent nicotine alters dendritic morphology in the bed nucleus of the stria terminalis.

Adolescent nicotine increases dendritic elaboration in several areas associated with the extended amygdala. It also increases anxiety-like behavior in adulthood. An unresolved question is whether adolescent nicotine alters dendritic structure in the bed nucleus of the stria terminalis (BNST), which may contribute to altered anxiety-like behavior. To investigate this possibility, adolescent male Sprague-Dawley rats were administered nicotine (0.5mg/kg/day) 3 days a week for 2 consecutive weeks, starting at postnatal day P (32). 17 days following the end of dosing, brains were processed for Golgi-Cox staining, and neurons were digitally reconstructed in three dimensions. Animals previously treated with nicotine exhibited an increase in the total number of branches and total length of dendrites on BNST neurons. Sholl analysis revealed an increase in the number of intersections with concentric spheres, increased amount of dendritic material within concentric spheres, and an increase of dendritic branching within concentric spheres occurring between 20 and 300 µm from the soma in dendrites. Collectively, our results show that adolescent nicotine alters dendritic structure (by triggering new branch growth), and, by inference, connectivity of the BNST, which may contribute to alterations in behavior induced by adolescent nicotine.

Androgen receptor immunoreactivity in forebrain axons and dendrites in the rat.

As members of the steroid receptor superfamily, androgen receptors (ARs) have been traditionally identified as transcription factors. In the presence of ligand, ARs reside in the nucleus, where, upon ligand binding, the receptors dimerize and bind to specific response elements in the promoter region of hormone-responsive genes. However, in this report, we describe the discovery that ARs are also present in axons and dendrites within the mammalian central nervous system. AR expression in axons was identified in the rat brain at the light microscopic level using two different antibodies directed against the N terminus of the AR protein and nickel intensified 3'-3'-diaminobenzidine, and also using fluorescence methods and confocal microscopy. This distribution was confirmed at the ultrastructural level. In addition, AR immunoreactivity was identified in small dendrites at the ultrastructural level. AR-immunoreactive axons were observed primarily in the cerebral cortex and were rare in regions where nuclear AR expression is abundant. The observation that ARs are present in axons and dendrites highlights the possibility that androgens play an important and novel extra-nuclear role in neuronal function.

Quantitation of synaptic density in the septal nuclei of young and aged Fischer 344 rats.

Synaptic density in the medial and lateral septal nuclei was examined in 3 and 24-28 months of age Fischer 344 rats. The lateral nucleus had a higher synaptic density than the medial region in both age groups. There were no statistically significant differences in synapse density in either region as a function of age, but the data suggested a subpopulation of aged animals which did show an age-related decline in synaptic density in the lateral, but not medial area of the septum. These data indicate that sample size may be an important variable in assessing possible age-related differences in synaptic density, since a broad range of values, some significantly below the range of young animals, exists in the aged brain.

Organization of the septal region in the rat brain: a Golgi/EM study of lateral septal neurons.

The combined Golgi/electron microscope (EM) technique was used to analyze the fine structure and synaptic organization of the various types of neurons in the rat lateral septum (LS), i.e., in the dorsolateral (LSd), intermediolateral (LSi), and ventrolateral (LSv) nuclei of the septal complex. Two characteristic cell types were observed in the LSd: type I with thick, short dendrites densely covered with short spines, and type II with longer and thinner dendrites exhibiting fewer but longer spines. This latter type was by far the most frequently impregnated cell type in the LSd and was also present in the LSi. Synaptic contacts on spines of either cell type were asymmetric; the majority of the presynaptic boutons contained clear round synaptic vesicles. Occasionally terminals were found that contained both clear and dense-core vesicles. Typical fusiform neurons with a low number of spines and rather long dendrites, sometimes invading other LS nuclei, were found in the LSi. The LSv contained numerous small neurons with small dendritic fields. A relatively large number of terminals with dense-core vesicles were found to establish synaptic contacts with identified LSv neurons. The morphological heterogeneity of LS neurons is discussed with regard to other studies on afferent and efferent fiber systems as well as immunohistochemical studies of this particular region of the septal complex.

Fine structure of rat septohippocampal neurons: II. A time course analysis following axotomy.

Previous light microscopic immunocytochemical studies with antibodies against transmitter-synthesizing enzymes have suggested that septohippocampal neurons undergo retrograde degeneration following transection of their axons by cutting the fimbria-fornix. However, a fine-structural analysis of the degeneration process in these cells is lacking so far. Here we have identified septohippocampal neurons by retrograde tracing with Fluoro-Gold. Thereafter, the fimbria-fornix was transected bilaterally. Fine-structural changes in prelabeled septohippocampal neurons were then studied after varying survival times up to 10 weeks. Examination under the fluorescence microscope of Vibratome sections through the septal region revealed numerous retrogradely labeled cells after all survival times following axotomy. These neurons were then intracellularly injected with the fluorescent dye Lucifer Yellow in order to stain their dendritic arbor. Many cells were found after each survival time that displayed characteristics of septohippocampal neurons in control rats (see Naumann et al., J Comp Neurol 325:207-218, 1992). In addition, increasing with survival time, there were many shrunken neurons with a reduced dendritic arbor. Representative examples of both normal appearing and shrunken neurons were photoconverted for subsequent electron microscopic analysis. Relatively few signs of neuronal degeneration were found at each survival time analyzed. The majority of cells, including the heavily shrunken ones, displayed fine-structural characteristics of normal neurons. However, a few degenerating neurons and reactive glial cells were present in all survival stages. We conclude that axotomized septohippocampal projection neurons cease the expression of transmitter-synthesizing enzymes and shrink, but many more cells survive for extended periods of time without target-derived neurotrophic factor than was assumed in previous light microscopic studies.

Morphology of neurons in the basal forebrain nuclei of the rat: a Golgi study.

The neuronal cell types and their morphology in the nucleus basalis (NB), in the horizontal and vertical limbs of the diagonal band of Broca (NHL and NVL), and in the medial septal nucleus (MSN) were examined in Golgi-impregnated material. Cells appeared as multipolar or oligopolar and displayed a variable dendritic morphology; their somata varied considerably both in shape and size. The dendrites of most cells were restricted within nuclear boundaries, although occasionally neurons located near boundaries, particularly cells in NHL, extended dendritic arbors into neighboring areas. Axons were rarely seen, but when they were found they were generally not impregnated beyond the initial segment and displayed no apparent preferential direction. Three types of cells common to each of the 4 nuclear groups could be identified on the basis of soma shape and dendritic form. The first type included large multipolar neurons with triangular or polygonal perikarya and typically 3-5 dendrites emerging from the poles of each cell. These cells were especially numerous in NB, NHL, and NVL, but were much less frequent in MSN. The second type comprised medium-sized cells with round or oval somata and a small number, usually 2-3, of dendrites. They constituted a large percentage of the cell population in MSN, but were also encountered in NHL and NVL as well as in NB. The third type consisted of cells with fusiform or spindle-shaped somata with usually single dendrites emanating from each pole of the cell. A rare but distinct type of spindle-shaped neuron with dendrites bearing a rich complement of long and thin appendages was observed mainly in the ventral border of NHL. The present observations suggest that although the proportions and sizes of the 3 types of neurons vary between the 4 nuclei, neurons throughout the basal forebrain share common morphological characteristics.

Choline acetyltransferase-immunoreactive neurons and terminals in the rat septal complex: a combined light and electron microscopic study.

A monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme, was used to determine the morphological characteristics of cholinergic neurons and axon terminals within the rat septum. Light microscopy revealed numerous large fusiform or multipolar ChAT-immunoreactive neurons in the medial septal nucleus/diagonal band complex (MSDB). In contrast, virtually no immunostained cells were found in the lateral septum (Nc. septalis dorsalis and Nc. septalis lateralis). Fine immunostained fibers were most abundant close to the midline in the MSDB mainly following an ascending course. A few thin ChAT-immunoreactive fibers and terminallike pericellular punctate structures were observed in the inner part of the dorsal septal nucleus. Electron microscopy of ChAT-immunoreactive neurons revealed large cell bodies rich in cytoplasmic organelles. The cell nuclei regularly exhibited multiple invaginations of the nuclear membrane. Only rarely were terminals found that established synaptic contacts on the cell bodies of immunostained neurons. In contrast, numerous terminals formed synaptic contacts on immunoreactive dendrites. ChAT-immunopositive terminals were studied in thin sections from the MSDB and from the dorsal septal nucleus. In both regions they appeared as heavily immunostained vesicle-filled boutons that established symmetric and asymmetric synaptic contacts. In the dorsal septal nucleus immunostained terminals often showed a basketlike arrangement around immunonegative cell bodies. Our fine structural study provides evidence that cholinergic neurons in the MSDB are similar to cholinergic neurons in the basal nucleus and neostriatum, which have been described by other investigators. The presence of cholinergic synapses in the septal complex indicates that this region not only contains cholinergic projection neurons, but receives a cholinergic input itself.

Fine structure of rat septohippocampal neurons: I. Identification of septohippocampal projection neurons by retrograde tracing combined with electron microscopic immunocytochemistry and intracellular staining.

In this report the normal dendritic organization and fine structure of identified septohippocampal projection neurons is described as a prerequisite for a time course analysis of retrograde changes in these neurons following axotomy (see Naumann et al., J. Comp. Neurol. 325:219-242, 1992). Septohippocampal projection neurons were retrogradely labeled by injection of the fluorescent tracer Fluoro-Gold into the hippocampus. Next, retrogradely labeled cells in Vibratome sections of the medial septum/diagonal band complex were intracellularly stained with the fluorescent dye Lucifer Yellow (LY). Photooxidation of LY resulted in a stable electron-dense reaction product, which allowed us to study these double-labeled neurons by electron microscopy. Another series of sections containing retrogradely labeled neurons were immunostained for choline acetyltransferase (ChAT) or parvalbumin (PARV). In this way the fine structure of two different chemically characterized subpopulations of septohippocampal neurons could be compared with that of the LY-injected neurons. Intracellular filling of retrogradely labeled neurons with LY stained the cell body and the entire dendritic arbor. Essentially, three classes of neurons could be distinguished, i.e., bipolar cells, multipolar neurons, and an intermediate group. All these neurons displayed smooth, often varicose dendrites lacking spines. Mainly located close to the midline, there was a group of cells with only very few if any LY-stained dendrites. In the electron microscope, the double-labeled neurons were easily identified by numerous electron-dense lysosomes associated with transported Fluoro-Gold and the diffuse reaction product resulting from photooxidation. They displayed fine-structural characteristics as previously described for cholinergic neurons. In fact, our fine-structural analysis of ChAT-positive Fluoro-Gold-labeled neurons, but also of back-filled PARV-positive cells, gave very similar results. All these neurons had infolded nuclei, abundant cytoplasmic organelles, and a few axosomatic synapses. Thus, a plain electron microscopic study does not allow one to distinguish between subpopulations of septohippocampal projection neurons.

Organization of the septal region in the rat brain: cholinergic-GABAergic interconnections and the termination of hippocampo-septal fibers.

This study deals with two characteristic cell types in the rat septal complex i.e., cholinergic and GABAergic neurons, and their synaptic connections. Cholinergic elements were labeled with a monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme. Antiserum against glutamate decarboxylase (GAD), the GABA synthesizing enzyme, was employed to identify GABAergic perikarya and terminals, by using either the peroxidase-antiperoxidase (PAP) technique or a biotinylated second antiserum and avidinated gold or ferritin. With these contrasting immunolabels we have studied the cholinergic-GABAergic interconnections in double-labeled sections of intact septal regions and the GABAergic innervation of medial septal area cholinergic neurons in sections taken from animals 1 week following lateral septal area lesion. In other electron microscopic experiments we have studied cholinergic and GABAergic neurons in the septal complex for synaptic contacts with hippocamposeptal fibers, which were identified by anterograde degeneration following fimbria-fornix transection. Our results are summarized as follows: (1) GAD-positive terminals form synaptic contacts on ChAT-immunoreactive dendrites in the medial septum/diagonal band complex (MSDB), (2) surgical lesion of the lateral septal area resulted in a dramatic decrease of the number of GABAergic boutons on MSDB cholinergic neurons, (3) cholinergic terminals establish synaptic contacts with GAD immunoreactive cell bodies and proximal dendrites in the MSDB as well as in the lateral septum (LS), (4) degenerated terminals of hippocampo-septal fibers were mainly observed in the LS, where they formed asymmetric synaptic contacts on dendrites of GABAergic neurons and on nonimmunoreactive spines. We did not observe degenerated boutons in contact with ChAT-positive dendrites or cell bodies in the MSDB. From these results and from data in the literature we conclude that excitatory hippocampo-septal fibers activate GABAergic cells, and as yet unidentified spiny neurons in the LS, which may control the discharge of medial septal cholinergic neurons known to project back to the hippocampal formation.

Cholinergic neurons in the rat septal complex: ultrastructural characterization and synaptic relations with catecholaminergic terminals.

Physiological and pharmacological studies have suggested that catecholamines modulate cholinergic neurons in the medial septal and diagonal band nuclei (i.e., the septal complex). Thus, the ultrastructural morphology of neurons containing choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine, and their relation to catecholaminergic terminals exhibiting immunoreactivity for the catecholamine synthesizing enzyme tyrosine hydroxylase (TH) were examined in the rat septal complex. Dual immunoautoradiographic and peroxidase anti-peroxidase labeling methods were used to simultaneously localize antibodies raised in rabbits against TH and from rat-mouse hybridomas against ChAT in single sections. At least two types of perikarya with ChAT-immunoreactivity (ChAT-I) were observed. The first type were large (20-30 microns), elongated or round, and contained a small indented nucleus with an abundant cytoplasm and an occasional lamellar body. The second type was also either ovoid or round but was medium-sized (15-20 microns) and contained a larger indented nucleus and a smaller amount of cytoplasm than the first type. Both types of perikarya as well as dendrites with ChAT-I were surrounded by astrocytic processes apposed to most of their plasmalemmal surfaces. The distribution and types of terminal associations (i.e., asymmetric synapses, symmetric synapses and appositions which lacked a membrane specialization in the plane of section analyzed) with ChAT-labeled perikarya and dendrites were quantitatively evaluated. The majority (68% of 197) of the presynaptic terminals were unlabeled; the remaining terminals were immunoreactive for TH (25%) or ChAT (7%). All three types of terminals contacted primarily the shafts of small dendrites and more rarely ChAT-labeled perikarya and large dendrites. ChAT-labeled terminals: (1) formed associations with unlabeled perikarya and dendrites (31% of 176); (2) formed associations with perikarya and dendrites with ChAT-I (7%); (3) contacted the same unlabeled perikarya and dendrite as a TH-containing terminal (21%); (4) were in apposition to TH-labeled terminals (25%); or (5) were either in apposition to unlabeled or ChAT-labeled terminals or lacked associations with any processes. The majority of associations formed by the terminals with ChAT-I were on the shafts of small dendrites. Moreover, most of the associations formed were either symmetric synapses or appositions not separated by astrocytes in the plane of section analyzed. These findings provide cellular substrates in the septal complex (1) for sparse synaptic input relative to astrocytic investment of cholinergic neurons and (2) for direct synaptic modulation of cholinergic and non-cholinergic neurons by catecholamines and/or acetylcholine. These findings have direct relevance to catecholaminergic-cholinergic interactions and to the neuropathological basis for Alzheimer's disease.

Juxtacapsular nucleus of the stria terminalis of the adult rat: extrinsic inputs, cell types, and neuronal modules: a combined Golgi and electron microscopic study.

This study unravels the microscopic organization of the juxtacapsular nucleus of the bed nuclei of the stria terminalis (Ju) by using silver impregnation and electron microscopic techniques. Examination of Golgi-impregnated specimens demonstrates that the Ju has precise boundaries primarily determined by a conical condensation of fibers of the stria terminalis (StT) around the nucleus. The internal capsule, ansa peduncularis, and medial forebrain bundle together with the StT provide extrinsic afferents to the neuropil of the Ju. Two main neuron types are found in the Ju: interneurons (including basket and neurogliaform cells) and projection neurons (bipolar and small pyramidal cells). The bipolar cell type accounts for about 80% of the sampled neurons. Short-axon neurons located within the dorsal part of the Ju send descending fibers that appear to terminate on the bipolar neurons, suggesting the existence of vertically oriented functional units within the nucleus. With the electron microscope, Ju neurons are seen in clusters of two or three neurons coupled by gap junctions. The neuropil contains numerous dendrites, axons, myelinated axons, and several types of synaptic interactions, including axospinous, axoshaft, and axosomatic. Within the neuropil, Ju neurons appear to be presynaptically modulated by axoaxonal interactions. The present findings suggest a model wherein bipolar neurons represent the output system of the Ju controlled by the interneurons, which would, in turn, be modulated by collaterals arising from the tributary fiber tracts. Additional neural interaction between Ju neurons utilizes gap junction-mediated electrotonic coupling.

Noradrenergic innervation of the developing and mature septal area of the rat.

The noradrenergic innervation of the developing and mature septal area of the rat was examined with light and electron microscopic immunocytochemistry using an antibody against dopamine-beta-hydroxylase. At birth, a small number of relatively thick noradrenergic fibers were found to innervate the lateral septum (mainly its intermediate part) and the nuclei of the vertical and horizontal limbs of the diagonal band of Broca. By postnatal day 7, a substantial increase in their density was observed. At this age some labeled fibers left the medial forebrain bundle and invaded the nucleus of the horizontal limb of the diagonal band. These fibers then ran in a ventrodorsal direction and innervated the nucleus of the vertical limb before entering the medial septum. Immunoreactive fibers were finer and more varicose than at birth. In the subsequent 2 weeks, the density of labeled fibers in the septal area was further increased. By postnatal day 21, the distribution pattern and density of the noradrenergic innervation appeared similar to the adult. In the adult, noradrenergic fibers exhibited more varicosities than in younger rats. Electron microscopic analysis revealed a low proportion (peaked at P7) of noradrenergic varicosities engaged in synaptic contacts throughout development. The overwhelming majority of these synapses were symmetrical, predominantly with small or medium-sized dendrites. The present findings provide the morphological basis for the functional interactions between noradrenergic afferents and neuronal elements in the septal area. The low proportion of synaptic contacts found in this study suggests that noradrenaline may exert its action in the septal area mainly through transmission by diffusion (volume transmission), as has been suggested for other areas of the developing and adult brain.

Experimental immune-mediated damage of septal cholinergic neurons.

Degeneration of cholinergic neurons in the medial septum and the diagonal band of Broca is a frequent neuropathological feature of Alzheimer's disease. To determine whether an immune process can injure these basal forebrain cholinergic neurons, we serially immunized guinea pigs with septal cholinergic hybrid cells (SN-56). Following immunization, a relatively selective damage of septal cholinergic neurons, reduction in septal choline acetyltransferase (ChAT) activity and decrease in acetylcholine release in hippocampus were detected. Serum IgG from guinea pigs immunized with SN-56 cells and stereotactically injected into the medial septal region of rats produced a loss of ChAT activity in the medial septum, frontal cortex and hippocampus, together with impairment of learning and long term spatial memory. These data suggest that relatively selective damage to septal cholinergic neurons can be caused by an immune-mediated process in experimental animals.

Serotonin1A receptors are expressed by a subpopulation of cholinergic neurons in the rat medial septum and diagonal band of Broca--a double immunocytochemical study.

The possible colocalization of 5-hydroxytryptamine1A receptors and choline acetyltransferase in the same neurons of the medial septum and diagonal band of Broca was investigated using double immunocytochemical techniques, either on the same section or on adjacent thin sections of the rat brain. The presence of both antigens in the same neurons was demonstrated at the light and electron microscopic levels. The proportion of cholinergic neurons that express 5-hydroxytryptamine1A receptors was similar in the different parts of the septal complex (around 25%). By contrast, the proportion of 5-hydroxytryptamine1A receptor-positive neurons also exhibiting choline acetyltransferase immunoreactivity was much higher (40-44%) in the dorsal and ventral groups of cholinergic cells, than in the intermediate group (18%). In line with the topographical distribution of cholinergic projections, this result points out the potential involvement of 5-hydroxytryptamine1A receptors in the control of the septohippocampal cholinergic projection by serotonin. This connection might be relevant to learning and memory, and in the appearance of age-dependent or neurodegenerative cognitive deficits, which have been shown to involve alterations in both the serotoninergic and the cholinergic systems.