An electrophysiological study of the neural projections from the hippocampus to the ventral pallidum and the subpallidal areas by way of the nucleus accumbens

The integrative role of the ventral striatum in transmitting signals from the hippocampus to the ventral pallidal and subpallidal areas was investigated in urethane-anaesthetized rats using an extracellular single-unit recording technique. Neurones of the nucleus accumbens were first activated by single-pulse stimulation of the ventral subiculum of the hippocampus. Further tests were made to investigate whether these accumbens neurones could be activated antidromically by stimulation of either the ventral pallidal or subpallidal areas. More than 4 times as many accumbens neurones, activated by hippocampal stimulation, responded antidromically to stimulation of subcommissural ventral pallidum than to stimulation of the sublenticular subpallidal area. This observation suggests that the hippocampus has preferential inputs to accumbens efferent neurones which project monosynaptically to the ventral pallidum. Spontaneously active neurones in the ventral pallidum and subpallidal area were inhibited by stimulation of the ventral subiculum of the hippocampus. These inhibitory responses were reduced when glutamic acid diethyl ester, a glutamate antagonist, was microinjected into the medial accumbens, apparently blocking the hippocampal-accumbens glutamatergic synapses to both the ventral pallidal-directed and the subpallidal-directed accumbens efferents. This evidence suggests that signals from the hippocampus reach ventral pallidal and subpallidal regions by way of the nucleus accumbens. The presence of a projection from ventral pallidal and subpallidal regions to the brainstem mesencephalic locomotor region further supports the hypothesis that limbic (e.g. hippocampus) can influence somatomotor activities by way of the nucleus accumbens and its efferent projection to ventral pallidal and subpallidal regions.     

Dual (excitatory and inhibitory) calretinin innervation of AMPA receptor-containing neurons in the rat lateral septum

A recent study demonstrated both an extrinsic and an intrinsic calretinin (CR) innervation of the rat septal complex and that a population of the extrinsic calretinin fibers is aspartate/glutamate-containing. The aim of this study was to determine which types (GluR1, GluR2/3, or both) of AMPA receptor-containing lateral septal area neurons are innervated by extrinsic and intrinsic CR neurons and whether the intrinsic CR cells are GABAergic. Light- and electron-microscopic single immunostaining for CR, GluR1, and GluR2/3, as well as light- and electron-microscopic-double immunostaining experiments for CR plus GluR1 and CR plus GluR2/3 were performed in the lateral septal area. Furthermore, the ″mirror″ colocalization technique was employed on consecutive vibratome sections of the septal complex to investigate whether the intrinsic septal CR neurons are GABAergic. The results are summarized as follows: (1) both GluR1- and GluR2/3-immunoreactive neurons are innervated by CR-containing fibers; (2) the majority of these synapses, observed mainly on the soma and, to a lesser extent, on proximal dendrites of AMPA receptor-containing neurons, represent asymmetric synaptic membrane specializations; (3) a minority of CR-containing axon terminals associated with both GluR1- and GluR2/3-immunoreactive neurons form symmetric contacts, predominantly on their soma; and (4) 93% of the lateral septal area CR cells are GABAergic. These observations indicate that both GluR1- and GluR2/3-containing lateral septal area neurons receive a dual intrinsic and extrinsic CR innervation. The former (intrinsic) CR boutons are GABAergic, while the latter form asymmetric synaptic contacts, are excitatory, and probably originate in the supramammillary area, since previous work has demonstrated that a population of supramammillo-septal fibers contain aspartate and/or glutamate. Received: 23 May 1997 / Accepted: 21 August 1997     

Changes with aging in the dopaminergic and noradrenergic innervation of rat neocortex

In normal aging, the mammalian cortex undergoes significant remodeling. Although neuromodulation by dopamine and noradrenaline in the cortex is known to be important for proper cognitive function, little is known on how cortical noradrenergic and dopaminergic presynaptic boutons are affected in normal aging. Using rats we investigated whether these two neurotransmitter systems undergo structural reorganization in aging, and if these changes correlated with cognitive loss. Young and aged rats were tested for cognitive performance using the Morris water maze. Following the behavioral characterization, the animals were sacrificed and the cortical tissue was processed for immunofluorescence using antibodies directed against tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) to detect and discriminate noradrenergic and dopaminergic varicosities. We observed a significant increase in dopaminergic varicosities in lamina V of the anterior cingulate cortex (ACC) of aged cognitively unimpaired rats when compared to young and aged-impaired animals. In laminae II and III of the ACC, we observed a significant decrease of dopaminergic varicosities in aged-impaired animals when compared to young or aged cognitively unimpaired animals. Changes in noradrenergic varicosities never reached statistical significance in any group or brain region. The data suggests that the remodeling of mesocortical dopaminergic fibers may participate in age-associated cognitive decline.     

Ultrastructural features of the serotonin innervation in adult rat hippocampus: an immunocytochemical description in single and serial thin sections

This study was aimed at characterizing the fine-structural features of the normal serotonin (5-HT) innervation in adult rat hippocampus, by means of electron microscopic immunocytochemistry with a polyclonal antiserum against 5-HT-glutaraldehyde-protein conjugate (donated by Michel Geffard, Bordeaux). Two hippocampal sectors were examined, at mid-level along the septo-temporal axis: CA3-a of Ammon's horn and crest of the dentate gyrus (DG-c). A large number of axonal varicosities (terminals) were sampled in single ultrathin sections, to achieve a statistically significant comparison of their size and of their relative frequency of synaptic specialization, junctional targets and juxtaposed elements, between the oriens and the radiatum layer of CA3-a, and the molecular and the polymorph layer of DG-c. In both CA3-a layers, the microenvironment of the immunostained terminals was also compared to that of a population of unlabeled varicosities randomly selected from the same micrographs. Moreover, 57 varicosities from the oriens and the radiatum layer of CA3-a were visualized in a long series of thin sections, allowing for their examination from end to end in 43 instances. As measured in single sections, hippocampal 5-HT varicosities were of comparable diameter (0.57 microns on the average) in the two anatomical sectors and four neuropil layers examined. As extrapolated stereologically to whole varicosities, the proportion making a synaptic membrane specialization (synaptic incidence) ranged from 18 to 33% (average of 24%), without statistically significant differences between the two sectors and four layers. The synaptic incidence determined directly from serial sections of CA3-a (18%) was nearly identical to that extrapolated from single sections (18.1% in the oriens and 19.5% in the radiatum layer). In both CA3-a and DG-c, the 5-HT varicosities showing a junctional complex were slightly larger than their non-junctional counterparts. In CA3-a, only dendritic shafts were targeted by synaptic 5-HT varicosities, whereas in DG-c there were also a few axo-spinous synapses. The microenvironment of CA3-a 5-HT varicosities differed markedly from that of randomly selected unlabeled varicosities, due to its much lower frequency of synaptic targets and higher frequency of juxtaposed axonal varicosities, at least in the radiatum layer. In all four layers examined, other axonal varicosities were indeed the most frequently encountered neuronal element in the immediate vicinity of immunostained 5-HT varicosities. Neurites and dendritic shafts were also common, but dendritic spines (4%) were relatively infrequent.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructural study of ascending projections to the lateral mammillary nucleus of the rat

Summary We examined the synaptic organization of ascending projections from the pars ventralis of the dorsal tegmental nucleus of Gudden (TDV) and the laterodorsal tegmental nucleus to the lateral mammillary nucleus (LM). The LM neuropil consists of terminals containing pleomorphic synaptic vesicles and forming symmetric synaptic contact, and terminals containing round synaptic vesicles and forming asymmetric synaptic contact. They make up 63% and 37%, respectively, of all axodendritic terminals. All axosomatic terminals contain pleomorphic vesicles and make symmetric contact. Following injection of WGA-HRP into the TDV, many anterogradely labeled terminals and retrogradely labeled cells are found in the LM. Labeled terminals contact mainly proximal (more than 2 m diameter) and intermediate (1–2 m diameter) dendrites. Serial ultrathin sections of the LM show that 55% of axosomatic terminals are labeled anterogradely. Following injection of WGA-HRP into the laterodorsal tegmental nucleus, many anterogradely labeled terminals are found in the LM, but no retrogradely labeled cells are present. Labeled terminals contact mainly distal (less than 1 m diameter) and intermediate dendrites as well as somata. In the LM neurons, 46% of axosomatic terminals are labeled anterogradely. All labeled terminals from these nuclei contain pleomorphic vesicles and make symmetric synaptic contact. These results indicate that almost all axosomatic terminals come from the TDV and the laterodorsal tegmental nucleus, which send inhibitory inputs to the lateral mammillary nucleus.     

Adrenergic innervation of noradrenergic locus coeruleus neurons. A dual labeling immunocytochemical study in the rat

By means of dual immunocytochemistry, synaptic associations between adrenergic terminals and noradrenergic neurons were directly demonstrated in the rat locus ceruleus (LC). It could be estimated that every adrenergic afferent contacts at least one noradrenergic dendrite in the nucleus. An adrenergic innervation of non-noradrenergic targets was also evidenced. These data add to our knowledge on the synaptic circuitry by which activation of the adrenergic input could affect central mechanisms known to be influenced by LC neurons.     

Serotonergic innervation of the dorsal horn of rat spinal cord: light and electron microscopic immunocytochemical study

Summary The ultrastructure of serotonergic projections to the dorsal horn of the rat spinal cord has been investigated, using a highly specific polyclonal antiserum. The highest concentrations of immunoreactive profiles were found in lamina I and the outer part of lamina II (IIo). Intermediate concentrations were found in laminae III and IV, while the inner part of lamina II (IIi) was almost devoid of immunoreactivity. Whereas 60% of the profiles show at least one varicosity studded with synaptic vesicles, only one-fifth of the latter contributes to classical synapses, the remaining profiles being devoid of a facing postsynaptic density. The results are compared with those in the literature and our own results relative to other regions of the cord. It is concluded that the pauci-synaptic projections to the dorsal horn could correspond to a diffuse influence of serotonin, the targets for which are determined by the corresponding serotonergic receptors.     

Ultrastructural spectrum of medulloblastoma with immunocytochemical correlations

Electron microscopy was used to examine 72 cases of medulloblastoma to better characterize the ultrastructural spectrum of this tumor. Twenty-four cases showed prominent neural differentiation. Twenty-three cases showed minimal (21) or no (2) recognizable neural differentiation, and the remainder of the cases (25) showed intermediate differentiation. All 42 cases tested stained for neuron-specific enolase, 28 for synaptophysin, and 12 for neurofilament protein. All cases showed strong reactivity for glial fibrillary acidic protein (GFAP) within reactive astrocytes. Three cases showed reactivity for GFAP within tumor cells. Medulloblastoma exhibits a broad spectrum of neural differentiation, with nearly all cases showing at least some degree of this change, and it universally exhibits participation of reactive astrocytes which can create a potential for diagnostic confusion.     

The cholinergic innervation of the visual thalamus: an EM immunocytochemical study

Summary In this study we demonstrate at the ultrastructural level that both the dorsal lateral geniculate nucleus (dLGN), the visual relay of the thalamus, and the perigeniculate nucleus (PGN), the visual segment of the thalamic reticular nucleus (TRN), are densely innervated by fibres with Choline-Acetyl-Transferase (ChAT) like immuno-reactivity. These axons make synaptic contacts with interneurones considered to be inhibitory, both in the PGN and within the synaptic glomeruli of the dLGN. In addition, Chat positive terminals form intra- and extraglomerular synapses with dendrites thought to arise from relay cells. We interpret these results as evidence for direct cholinergic modulation of both relay cells and inhibitory interneurones.     

Fine structures of nerve fibers with corticotropin-releasing factor-like immunoreactivity in the rat lateral septum

The fine structures of nerve fibers with corticotropin-releasing factor (CRF)-like immunoreactivity in the rat lateral septum were investigated by means preembedding immunoelectron microscopy. A number of CRF axon terminals formed synapses with cell bodies of non-immunoreactive septal neurons. They occasionally had broad terminal bulges whose subregions showed little or no immunoreactivity for CRF. CRF axon terminals were also in synaptic contact with non-immunoreactive dendrites or dendritic spines. Some dendrites with CRF were postsynaptic to non-immunoreactive axon terminals.     

An immunohistochemical study of neuronal populations containing neuropeptides or gamma-aminobutyrate within the superficial layers of the rat dorsal horn

Immunohistochemical techniques have been used to characterise a number of interneuronal types found within the superficial layers of the rat dorsal horn. Substance P, methionine-enkephalin, neurotensin, avian pancreatic polypeptide, somatostatin and glutamic acid decarboxylase-like immunoreactivities were found within discrete populations of cell bodies within layers I to III of the spinal cord after colchicine pretreatment. Each immunoreactive cell type was distinct in terms of the distribution of terminals, perikaryal size and position within the dorsal horn. Cell bodies containing substance P were predominantly within layers I, II and III, while surgical isolation of the dorsal horn to remove terminals of extrinsic origin indicated that the terminals of the local substance P-containing interneurones were present throughout layers I and II₀. Enkephalin-immunoreactive neurones were most numerous within layer II, with less frequently observed larger enkephalin immunoreactive neurones in layer I. Neurotensin-containing neurones were found in a fairly narrow layer along the II/III border, with dendritic arborisations extending into both layers II and III. Neurotensin-immunoreactive terminals occurred in two bands, a dense band within layer II, and a less dense one within layers I and II₀. Somatostatin-like immunoreactivity was found in occasional large neurones within layer II_i. Avian pancreatic polypeptide immunoreactivity was localized to neurones within layers I, II and III, as was terminal immunoreactivity. Glutamic acid decarboxylase-containing neurones were large and were found predominantly within layers I, II ₀ and on the II/III border, where terminal immunoreactivity was also concentrated. Hemisection combined with dorsal root section indicated that the majority of these immunohistochemically identifiable neurones were interneurones.The interconnections and relations of these neurones to dorsal horn projection neurones are considered in the light of the reported analgesic properties of a number of these substances when applied directly to the spinal cord.     

Heterogeneity in the occurrence of a subset of autoantibodies to glutamic acid decarboxylase in autoimmune polyendocrine patients with islet cell antibodies

Glutamic acid decarboxylase-65 (GAD-65) is a major target for autoantibodies and autoreactive T cells in patients with insulin-dependent diabetes mellitus (IDDM). Autoantibodies to GAD are also found in patients with stiff man syndrome (SMS) or polyendocrine autoimmunity (PE). The epitope specificities of autoantibodies to GAD in IDDM and SMS have been well documented, but the locations of autoantibody epitopes of GAD in PE patients have not been mapped. Thus, the properties of anti-GAD antibodies in PE patients (with or without diabetes) were investigated. The ability of PE serum antibodies to inhibit the binding of the mouse monoclonal antibody, GAD-6, to native GAD in ELISA was determined. For PE patients without diabetes, levels of inhibition of GAD-6 binding ranged from 0% to almost 70% and were unrelated to the level of binding of serum antibodies to GAD (P = 0.351) or to the functional affinities of these antibodies. This suggests differences in the epitope specificities of anti-GAD antibodies in different patients. Levels of inhibition were also unrelated to clinical condition. SMS antibodies showed similar levels of inhibition of GAD-6 binding. Similar analysis was applied to PE patients with diabetes and levels of inhibition of GAD-6 binding to GAD were determined. These ranged from 0% to 80%, and levels of inhibition were similar in samples taken before or after diabetes onset. There was no significant difference between anti-GAD antibodies from PE patients with or without diabetes in the range of abilities to inhibit GAD-6 binding to GAD, although the highest levels of inhibition were given by sera from non-diabetic patients. This raises the possibility of differential expression of subsets of anti-GAD antibodies in progressive versus slow or non-progressive anti-islet autoimmune responses. Serum antibodies of PE and SMS patients did not inhibit the binding of antibodies specific for the extreme C-terminus of GAD, indicating that this is not the site of the epitopes for the patients' antibodies or for GAD-6.     

The C-terminal glycopeptide of propressophysin potentiates excitatory transmission in the rat lateral septum

Effects of peptides synthesized from the same precursor as vasopressin, i.e. the C-terminal 39-amino acid long glycopeptide and neurophysin II, were investigated for biological activities in electrophysiological experiments in brain slices of the rat lateral septum. These slices contained the glycopeptide as the predominant form and a fragment of it, amino acid sequence 22-39, as a minor form (8% of the glycopeptide 1-39), as shown by high performance liquid chromatography of extracts and by radioimmunoassay. None of the peptides, neurophysin II, the glycopeptide 1-39 and the fragment 22-39, tested in a concentration of 10(-12) M, had measurable effects on the resting membrane potential of the neurons. The glycopeptide and the fragment 22-39, however, increased, in some cells, for tens of minutes the excitatory postsynaptic potentials evoked in these neurons by stimulation of the fimbria fibers. The increase in input resistance, seen in many septal neurons treated with either of the peptides was not correlated with the excitatory postsynaptic potential increase. Neurophysin II affected neither the excitatory postsynaptic potentials nor the input resistance of the neurons. It is concluded that the glycopeptide 1-39 and the fragment 22-39 possess biological activities amongst which the facilitation of excitatory amino acid transmission on lateral septum neurons. Therefore, these peptides derived from the vasopressin precursor may act in concert with vasopressin to establish facilitation of excitatory transmission in the brain.     

Organization of diencephalic and brainstem afferent projections to the lateral septum in the rat

Ascending diencephalic and brainstem afferents to the lateral septal column were studied by retrograde transport of horseradish peroxidase following microiontophoretic injections in the various subdivisions of the lateral septal area. Predominantly ispilateral cells, of which several coincide with reported monoaminergic cell groups, appeared in the preoptic area, several hypothalamic nuclei, periventricular nuclei, raphe nuclei, pontine area and medulla oblongata. It is concluded that there is a heterogenous monoaminergic input to the lateral septal area organized in a complicated pattern along the longitudinal axis of the lateral septum.     

Ultrastructural and immunocytochemical characterization of the cellular phenotype in primary adenoid liver tumours of the rat

The cellular phenotype of 34 primary adenoid liver tumours induced in rats with N-nitrosomorpholine was studied by immunocytochemical and electron microscopical methods in order to elucidate the histo- and cytogenesis of these tumours. Three types of ducts were distinguished in the adenoid liver tumours at the ultrastructural level being characterized as of hepatocellular, transitional and cholangiocellular phenotype. The transitional cells took an intermediate position between the hepatocellular and the cholangiocellular phenotype. Frequent features of the hepatocyte-like differentiation were large round nuclei with a dispersed chromatin, glycogen-associated ER complexes, peroxisomes and the formation of bile canaliculi. Evidence for the relationship to bile ductular cells was provided by the regular association with a basement membrane, the (inconstant) positive immunohistochemical reaction for cytokeratin polypeptide KA-4, a poorly developed ER and small mitochondria. An additional finding in the ducts with a transitional cellular phenotype was the selective accumulation of mast cells integrated into the epithelium. Intimate associations between cells of the hepatocellular, transitional and cholangiocellular phenotype were observed at the light and electron microscopic level. The results suggest that a transdifferentiation (metaplasia) from cells with a hepatocellular to those with a transitional or cholangiocellular phenotype takes place in many liver tumours.     

The pre- and postnatal development of the dopaminergic cell groups in the ventral mesencephalon and the dopaminergic innervation of the striatum of the rat

In the adult rat the striatum is a compartmentalized structure, which is reflected in the inhomogeneous distribution of dopamine. As a first step to test the hypothesis that dopamine plays an organizational role in the development of the striatum, the ontogeny of the dopaminergic system was studied in detail with immunocytochemical methods employing antibodies against dopamine. Rat embryos, fetuses, pups and adults were perfusion-fixed with glutaraldehyde on all prenatal days from E11 onward, postnatally on P2, P4, P6, P7, P8, P13, P14, P20, P21, and in adult age. On E13 the first dopaminergic cells are detected in the ventral prosencephalon. On E14 two dopaminergic cell groups are present in the ventral mesencephalon, and fibres of these cells reach the ventrolateral part of the ganglionic eminence. In the next two days both the cell groups and their projections rapidly increase in size. On E17 the afferent dopaminergic fibres to the striatum become aligned and form huge bundles that are closely associated with the fascicles of the internal capsule. Rostrally, the development of the striatal dopaminergic innervation shows a clear ventrolateral to dorsomedial gradient, whereas more caudally the dopaminergic fibres innervate the striatum from a ventromedial position. The lateral parts of the otherwise compact mesencephalic cell groups consist of loosely arranged cells. From E17 onward these cells become arranged into a dorsal and a ventral group. Just before birth, on E21, the primordia of the dopaminergic cell groups in the substantia nigra pars compacta and pars reticulata can be observed. On E19 several centres with extensive fibre ramifications along the dorsolateral margin of the caudate putamen represent the first signs of the inhomogeneous distribution of dopaminergic fibres in the dorsal striatum seen during the next two weeks. In the following pre- and postnatal days these so-called dopaminergic "patches" also appear more medially. By the third postnatal week most of the patches are no longer detectable, and only the most dorsolaterally located ones, i.e. in the region where they first were detected on E19, remain visible through to the adult stage. Prenatally, no varicosities can be observed in the dopaminergic fibres. The first varicosities appear after birth. Their number increase rapidly during the first and second postnatal weeks and reaches near adult levels on P20. The development of the striatal dopaminergic innervation, and that of the "patches" in particular, is discussed in relation to the development of the mesencephalic dopaminergic cell groups.(ABSTRACT TRUNCATED AT 400 WORDS)