Distribution of [3H]cholecystokinin octapeptide binding sites in the hippocampal region of the rat brain as shown by in vitro receptor autoradiography

The distribution of binding sites for the neuropeptide cholecystokinin octapeptide in the rat hippocampal region was studied by using quantitative in vitro receptor autoradiography. Biochemical analysis of [3H]cholecystokinin octapeptide binding to tissue sections of the hippocampal region showed it to be of high affinity, to be saturable and approximately 50% specific at saturating concentrations. The binding of [3H]cholecystokinin octapeptide to hippocampal sections was dose-dependently blocked by cholecystokinin octapeptide, cholecystokinin and by pentagastrin. The autoradiographic analysis showed high densities of [3H]cholecystokinin octapeptide binding sites in the hilus of the area dentata, the outer three layers of the retrosplenial area and the presubiculum, layer 3 of the medial, but not the lateral, entorhinal area and the deep and superficial parts of layer 1 and 2, respectively of both the medial and the lateral entorhinal area. Medium binding densities were found in the parasubiculum and remaining layers of the entorhinal area and low densities occurred in the subiculum and in all subfields of Ammon's horn. The angular bundle and fornix-fimbria lacked specific [3H] cholecystokinin octapeptide binding sites. A very similar pattern of binding densities was found for [3H]pentagastrin. Comparisons of the cholecystokinin octapeptide receptor distribution with the cholecystokinin octapeptide innervation of the hippocampal region suggest that there exists a relatively good concordance in some hippocampal subfields such as the presubiculum and the entorhinal area between binding sites for [3H]cholecystokinin octapeptide and cholecystokinin-immunoreactive afferent input.     

The distribution of serotonin binding sites in the hippocampal region of the rat brain. An autoradiographic study

The distribution of serotonin binding sites was studied in the rat hippocampal region by using contact-film autoradiography after in vitro incubations of brain sections with 5-[3H]hydroxytryptamine, [3H]spiperone, and [3H]ketanserin, respectively. Biochemical studies of the 5-[3H]hydroxytryptamine binding to sections cut through the hippocampal region showed that at saturating concentrations of 5-[3H]hydroxytryptamine (2-2.5 nM) the specific binding was at least 50% of the total. The 5-[3H]hydroxytryptamine binding sites were found to be heterogeneously distributed within the hippocampal region with the highest densities present in the following parts: layers I and II and layers IV through VI of the entorhinal area, the radial layer of the subiculum and subfield CA1 of the Ammon's horn and the molecular layer of the area dentata. Moderate to low densities of binding was observed in layer III of the entorhinal area, the pre- and parasubiculum, the stratum pyramidale of the Ammon's horn, and the granular cell layer of the area dentata. Removal of the 5-hydroxytryptamine nerve terminals by systemic injections of the 5-hydroxytryptamine neurotoxin parachloroamphetamine resulted in no detectable reductions of 5-[3H]hydroxytryptamine binding in any brain region. Lesions of hippocampal cell bodies by intrahippocampal injections of ibotenic acid prevented the binding of 5-[3H]hydroxytryptamine within the area of the cell loss. Comparisons between the distribution of 5-hydroxytryptamine immunoreactive nerve terminals and the 5-[3H]hydroxytryptamine binding sites showed that in some areas of sparse 5-hydroxytryptamine innervation the 5-[3H]hydroxytryptamine binding was close to background (e.g. the pyramidal cell layer, the stratum lucidum) whereas in areas with little 5-[3H]hydroxytryptamine binding (e.g. layer III of the lateral entorhinal area, the presubiculum) a very dense 5-hydroxytryptamine innervation was found. The hippocampal 5-[3H]hydroxytryptamine binding was displaced neither by ketanserin (1 microM) nor by spiperone (1 microM), two drugs that bind to cortical 5-hydroxytryptamine2 receptors in the rat brain. Furthermore, the pattern of hippocampal [3H]spiperone binding differed considerably from that of 5-[3H]hydroxytryptamine. The [3H]ketanserin binding in the hippocampal region did not exceed background levels, except in the hilus of area dentata in the ventral hippocampus and entorhinal layer VI at the same level, where moderate binding was found.(ABSTRACT TRUNCATED AT 400 WORDS)     

The substance P innervation of the rat hippocampal region

Summary The distribution of substance P (SP) immunoreactive nerve cell bodies and preterminal processes was studied in the rat brain by using several anti-SP-antibodies in combination with immunohistochemical techniques. In normal rats and in rats pretreated with colchicine, SP immunoreactive preterminal processes were found in the hippocampal region, but SP positive cellbodies could be detected only after colchicine pretreatment. Medium-sized to large, multipolar cells immunoreactive for SP were found in stratum oriens of the hippocampal subfield CA3 and in the hilus of the area dentata. Medium-sized to small, round or fusiform cells were detected in the pyramidal layer of the ventral subiculum and in layers III–VI of the ventral entorhinal area. The SP stained preterminal processes were of two types. Numerous fine, varicose axons were stained in different parts of Ammon's horn, while in the retrohippocampal structures, the SP immunoreactivity was present in small distinctly stained puncta. These frequently formed pericellular arrangements around unstained cells, indicative of axosomatic contacts between SP terminals and cells in the hipocampus. In Ammon's horn, the densest SP innervation was found in strata oriens, radiatum and moleculare of subfields CA3a and CA2. Scattered fibers were also present in the stratum oriens of CA3a-c and in the hilus, in particular at ventral levels. In retrohippocampal structures, the SP innervation predominated in the deep pyramidal layer of the subiculum, the second layer of the presubiculum and in layers VI and IV of the medial and lateral entorhinal area. Many of these terminals may arise from local interneurons as well as from sources outside the hippocampal region.Taken together, these studies demonstrate a far more extensive innervation by SP, or a closely related peptide, of the rat hippocampal region than was previously recognized. This suggests that SP may play an important role in neurotransmission within the hippocampal region.Stephen Davies was supported by Travel grants from the Wellcome Trust and the Gurantors of Brain.     

Distribution of neuropeptide Y receptors in the rat hippocampal region

The distribution of binding sites for neuropeptide Y (NPY) was studied in the rat hippocampal region by using [3H]NPY together with quantitative in vitro receptor autoradiography. The highest density of specifically bound [3H]NPY was found in regio superior and regio inferior of Ammon's horn. Within these fields, stratum oriens, stratum pyramidale and stratum radiatum harboured the highest densities of [3H]NPY binding while stratum moleculare was relatively poor in [3H]NPY binding sites. In area dentata, the highest density of [3H]NPY binding was found in the inner one third of the molecular layer. In the presubiculum and in the entorhinal area, the outer two layers were slightly more enriched in [3H]NPY binding sites than were the deep layers. In all hippocampal subfields a clear gradient of increased [3H]NPY binding was found at successively more ventral levels.     

Distribution of neurotensin receptors in the primate hippocampal region: a quantitative autoradiographic study in the monkey and the postmortem human brain

The distribution of [3H]neurotensin ([3H]NT) binding sites in the monkey and the postmortem human brain was studied by using quantitative in vitro receptor autoradiography. Biochemical experiments carried out on tissue sections of the monkey hippocampus showed that the binding of [3H]NT was saturable, reversible and of high specificity. The hippocampal [3H]NT binding was displaced by fragment NT 8-13 but not fragment NT 1-8 of the peptide. The anatomical analysis showed a highly heterogeneous distribution of [3H]NT binding sites within both the monkey and the human hippocampal region. In both species the highest density of [3H]NT binding sites was found in the presubiculum (rank order of binding density: layer 2 greater than 6 greater than 1 greater than 3, 4, 5 in both monkey and man) and the entorhinal area (monkey: layer 4 greater than 6 greater than 5 greater than 1 greater than 2 greater than 3; human: layer 1 = 2 greater than 5 greater than 3). The subiculum and Ammon's horn were relatively poor in [3H]NT binding sites in both species. In the area dentata the highest density of [3H]NT binding sites was found in the hilar region.     

Autoradiographic localization of dopamine D2 receptors in the rat brain using the new agonist [3H]N-0437

The selective dopamine D2 agonist [3H]N-0437 was used to label dopamine receptors in vitro in slide-mounted rat brain microtome sections. The characteristics of the binding of [3H]N-0437 to tissue section were similar to those observed previously in membrane preparations and indicated that this ligand labels sites with the properties of a dopamine D2 receptor. The regional distribution of these receptors was examined by autoradiography and quantified by computer-assisted microdensitometry. The highest densities of [3H]N-0437 sites were observed in the nucleus caudate-putamen, accumbens, olfactory tubercle, island of Calleja and the glomerular layer of the olfactory bulb. Lower densities of binding sites were seen in stratum griseum superficialis of the superior colliculus, substantia nigra pars compacta and area ventral tegmental, entorhinal cortex and in the molecular layer of the 9th and 10th lobules of the cerebellum. Very low densities were seen in the neocortex and hippocampal formation. The density of [3H]N-0437 binding sites in the rat striatum are higher than those observed with other dopamine D2 [3H]agonists and comparable to those seen with [3H]antagonists. [3H]N-0437 is a new useful tool for the anatomical localization of dopamine D2 receptors in brain.     

Autoradiographic localization of [3H]neurotensin-binding sites in the hippocampal region of the rat and primate brain

The distribution of binding sites for the neuropeptide neurotensin was studied in the hippocampal region of the rat, monkey and human brain by using the method of in vitro receptor autoradiography. Biochemical studies of [3H]neurotensin binding to homogenates or sections of the rat hippocampal region showed it to be saturable, reversible and of high specificity. Displacement studies showed that neurotensin-(1-13) and neurotensin-(8-13) were active, while neurotensin-(1-6) and (1-8) were inactive in blocking the specific binding of [3H]neurotensin to hippocampal sections. The autoradiographic studies showed a highly heterogeneous pattern of [3H]neurotensin binding in the hippocampal region: the highest density was present in the entorhinal area while little binding was found in the Ammon's horn. In the rat most of the [3H]neurotensin binding was found in layer II of the medial entorhinal area and in the parasubiculum, while the lateral entorhinal area contained fewer [3H]neurotensin-binding sites. The laminar distribution of binding remained the same throughout the longitudinal axis of the entorhinal area. The pattern of [3H]neurotensin binding in the monkey resembled that seen in the rat inasmuch as the medial was rich and the lateral entorhinal area was poor in [3H]neurotensin-binding sites. In the medial entorhinal area most binding was found in layers I-IV. Unlike in the rat, the hilus of the monkey contained moderate and the molecular layer of the area dentata few [3H]neurotensin-binding sites. In the human brain the outer three layers of both the medial and the lateral entorhinal area contained binding sites for [3H]neurotensin. Binding sites for [3H]neurotensin were found also in the parasubiculum and in the molecular layer of the area dentata of the human brain. The present autoradiographic studies show that the hippocampal region of the rat and primate brain is rich in binding sites for [3H]neurotensin, that a majority of these are situated in the entorhinal area and that despite some differences in the regional distribution of these binding sites within the hippocampal region, some principal similarities may exist between these species.     

Zinc-positive afferents to the rat septum originate from distinct subpopulations of zinc-containing neurons in the hippocampal areas and layers

The purpose of the present study was to examine whether zinc-positive and zinc-negative hippocampal neurons in rats differed with respect to their projections to the septum. By combining retrograde axonal transport of the fluorescent tracer Fluoro-Gold with histochemical demonstration of zinc selenide complexes in zinc-containing neurons after intraperitoneal injection of sodium selenite, we were able to visualize the distribution of retrogradely Fluoro-Gold labeled neurons and zinc-containing neurons in the same sections. After unilateral injection of Fluoro-Gold into the rat septum a few retrogradely labeled cells were observed in layer IV of the ipsilateral medial entorhinal area, and numerous labeled cells were observed mainly in the superficial layers of the ipsilateral subicular areas and throughout the CA1 and CA3 pyramidal cell layers, as well as in the contralateral CA3 pyramidal cell layer. Zinc-containing neurons were observed in layers IV–VI of the medial entorhinal area, layers II and III of the parasubiculum, layers II, III and V of presubiculum, and in the superficial CA1 and deep CA3 pyramidal cell layers. Cells double-labeled with Fluoro-Gold and zinc selenide complexes were primarily located in distal (relative to the area dentata) parts of the superficial CA1 pyramidal cell layer and distal parts of the deep CA3 pyramidal cell layer and in layers II and III of presubiculum. Only a very few double-labeled cells were seen in the contralateral CA3. The result demonstrates that the hippocampo-septal projection of rats is a mixture of zinc-positive and zinc-negative fibers. Where-as zinc-negative fibers originate from neurons throughout the hippocampal and retrohippocampal areas, zinc-positive fibers originate from distinct subgroups of zinc-containing cells in different areas and layers.     

Neurons and terminals in the retrohippocampal region in the rat''s brain identified by anti-gamma-aminobutyric acid and anti-glutamic acid decarboxylase immunocytochemistry

Summary The distribution of gamma-aminobutyric acid (GABA) containing nerve cells and terminals was studied at the light and electron microscopic levels in the retrohippocampal region of the rat by using anti-glutamic acid decarboxylase (GAD) and anti-GABA antibodies in immunocytochemistry. Large numbers of GAD and GABA stained cells were found in all retrohippocampal structures. At the ultrastructural level, the immunoreactivity against GABA and against the synthesizing enzyme GAD was localized to cytoplasmic structures, including loose clumps of rough endoplasmic reticulum, ribosomal arrays, outer mitochondrial surfaces and in axonal boutons.The GAD- and GABA-immunorective(-i) cells were found in all subfields of the retrohippocampal region (e.g., the subicular complex, the entorhinal area). Within the entorhinal area a slightly larger number of immunoreactive cells could be detected in layers II and III than in the other layers. In the subiculum, pre- and parasubiculum the GAD and GABA-i cells were present in relatively large numbers in all layers, except the molecular layer, which contained only a small number of GABA cells. Within the entorhinal area, GAD and GABA stained cells ranged in size from small (13 m in diameter) to large (22 m in diameter). A large number of different morphological classes of cells were found, except pyramidal and stellate cells. In the pre- and parasubiculum, on the other hand, the GABA cells were generally small to medium in size and morphologically more homogeneous than in the subiculum and entorhinal area.The entire retrohippocampal region was densely innervated by GABA preterminal processes, with little variation in the regional density of innervation. Within the entorhinal area, presubiculum and subiculum, a clear difference was found in the laminar pattern of innervation. In all three subfields the densest innervation was in layer II. In the entorhinal area both GAD- and GABA-i axons form palisades of fibers around the somata of neurons, which are tightly packed together in this layer. In the electron microscope both GAD-i and GABA-i were demonstrated in these axons. Axosomatic synaptic contacts were common between axons and the stellate neurons and other cells of this layer. Layers IV and VI appeared less dense in GAD-i terminals but appeared more densely innervated than layers III and V. The lamina dessicans was relatively poor in GAD-i. In the subiculum and presubiculum, as well as all other subfields of the hippocampal region, the innervation is dominated by axo-somatic innervation of layer II cells. The outer third of the molecular layer was more densely innervated than the inner part. Taken together, the present study has shown that the retrohippocampal region is rich in GABAergic neurons as well as axon terminals, some of which form numerous synapic contacts with cells of the region. GABAergic neurotransmission is an important mechanism in retrohippocampal circuits not only for the resident interneuronal population but in the surround as well.     

Terminal distribution of projections from the retrosplenial area to the retrohippocampal region in the rat, as studied by anterograde transport of biotinylated dextran amine

The terminal distribution of projections from the retrosplenial area to the retrohippocampal region was examined in the rat with anterograde transport of biotinylated dextran amine. Projections from the retrosplenial granular area (RSG) to the retrohippocampal region terminate predominantly ipsilaterally in layers I, III, V and VI of the presubiculum, layers I and IV–VI of the parasubiculum, the molecular and pyramidal cell layers of the subiculum, and layers I, III, V and VI of the entorhinal area. On the other hand, projections from the retrosplenial agranular area (RSA) terminate predominantly ipsilaterally in layers I and III of the presubiculum and layers V and VI of the entorhinal and perirhinal areas, and ipsilaterally in layers IV–VI of the parasubiculum. The results show that projections from the RSG to the retrohippocampal region are as massive as those from the RSA, and that each retrosplenial area has distinct projection fields in the retrohippocampal region. This suggests that each retrosplenial area may play some distinct functional roles in memory and learning processes such as spatial behavioral learning.     

Projections from the presubiculum and the parasubiculum to morphologically characterized entorhinal-hippocampal projection neurons in the rat

The relations between the inputs from the presubiculum and the parasubiculum and the cells in the entorhinal cortex that give rise to the perforant pathway have been studied in the rat at the light microscopical level. Projections from the presubiculum and the parasubiculum were labeled anterogradely, and, in the same animal, cells in the entorhinal cortex that project to the hippocampal formation were labeled by retrograde tracing and subsequent intracellular filling with Lucifer Yellow. The distribution and the number of appositions between the afferent fibers and hippocampal projection neurons in the various layers of the entorhinal cortex were analyzed. The results show that layers I–IV of the entorhinal cortex contain neurons that give rise to projections to the hippocampal formation. The morphology of these projection neurons is highly variable and afferents from the presubiculum and the parasubiculum do not show a preference for any specific morphological cell type. Both inputs preferentially innervate the dendrites of their target cells. However, presubicular and parasubicular projections differ with respect to the layer of entorhinal cortex they project to. The number of appositions of presubicular afferents with cells that have their cell bodies in layer III of the entorhinal cortex is 2–3 times higher than with cells in layer II. In contrast, afferents from the parasubiculum form at least 2–3 times as many synapses on the dendrites of cells located in layer II than on neurons that have their cell bodies in layer III. Cells in layers I and IV of the entorhinal cortex receive weak inputs from the presubiculum and parasubiculum. Not only is the presubiculum different from the parasubiculum with respect to the distribution of projections to the entorhinal cortex, they also differ in their afferent and efferent connections. In turn, cells in layer II of the entorhinal cortex differ in their electrophysiological characteristics from those in layer III. Moreover, layer II neurons give rise to the projections to the dentate gyrus and field CA3/CA2 of the hippocampus proper, and cells in layer III project to field CA1 and the subiculum. Therefore, we propose that the interactions of the entorhinal-hippocampal network with the presubiculum are different from those with the parasubiculum.     

Autoradiographic localization of D1 and D2 dopamine receptors in the human brain

The distribution of dopamine D1 and D2 receptors in several human brain regions was investigated using autoradiography with the radioligands [3H]SCH 23390 and [3H]spiroperidol. The highest densities of both dopamine receptor types are seen in the nucleus caudatus, putamen and nucleus accumbens. Whereas the density of the D2 receptors is similar in the two segments of the globus pallidus, the pars medialis of the globus pallidus contains a three-fold higher concentration of D1 receptors than the pars lateralis. D1 and D2 receptors are present in the amygdala and substantia nigra. Both receptor types are absent in the cerebellum. The thalamus contains low densities of D1 receptors but no D2 receptors. Only D2 receptors are seen in the anterior lobe of the pituitary gland. The whole cerebral cortex is rich in D1 receptors, while D2 receptors, in low concentrations, are confined to the entorhinal area and cingulate cortex.     

The distribution of cholecystokinin receptors in the vertebrate brain: species differences studied by receptor autoradiography

The regional distribution of cholecystokinin receptors in the brain of several vertebrate species including pigeon, rat, mouse, guinea-pig, cat, monkey and human was studied by in vitro auto-radiography using [125I] Bolton-Hunter cholecystokinin-octapeptide as a ligand. Cholecystokinin receptors presented marked species differences in several brain regions particularly in the laminar distribution of the cerebellar and cortical layers, in the hippocampal formation and in the basal ganglia. No binding was observed in the cerebellum of pigeon and rat, while intermediate to high densities of binding was observed in the cerebellum of the other species studied. In the pigeon external striatum, corresponding to the mammalian neocortex, low densities of cholecystokinin binding sites were observed. In contrast, mammalian neocortex was rich in cholecystokinin binding sites. However, the lamination was different from species to species, with high densities predominating in lamina IV in rat, mouse and guinea-pig cortex, in lamina II and VI in cat and monkey cortex and in lamina V in human cortex. Another area presenting important species differences was the hippocampal formation. The pigeon hippocampus showed intermediate densities of cholecystokinin receptors. In the mammals studied, very different patterns and densities of cholecystokinin binding sites were observed in the hippocampal formation. The basal ganglia were labelled in all species examined except the mouse. While biochemical and pharmacoganglia were labelled in all species examined except the mouse. While biochemical and pharmacological studies have shown that cholecystokinin receptors in different species present comparable characteristics, our autoradiographic investigations indicate that cholecystokinin binding sites are expressed differentially in several brain regions of different vertebrate species. The present autoradiographic study provides a morphological basis for further analysis on the expression and functionality of cholecystokinin receptors.     

Localization of preprogalanin mRNA in the monkey hippocampal formation.

The existence of neurons expressing preprogalanin mRNA in the monkey hippocampal formation was demonstrated using in situ hybridization of a radio-labelled cRNA probe transcribed from human preprogalanin cDNA. Specific hybridization occurred in neurons of the hilus of the dentate gyrus, fields CA1-3 in Ammon's horn, subiculum, presubiculum, parasubiculum and occasionally in neurons of the entorhinal cortex. These findings suggest that galanin is synthesized by neurons intrinsic to the monkey hippocampal formation.     

Dopamine has bidirectional effects on synaptic responses to cortical inputs in layer II of the lateral entorhinal cortex

Dopaminergic modulation of neuronal function has been extensively studied in the prefrontal cortex, but much less is known about its effects on glutamate-mediated synaptic transmission in the entorhinal cortex. The mesocortical dopamine system innervates the superficial layers of the lateral entorhinal cortex and may therefore modulate sensory inputs to this area. In awake rats, systemic administration of the dopamine reuptake inhibitor GBR12909 (10 mg/kg, ip) enhanced extracellular dopamine levels in the entorhinal cortex and significantly facilitated field excitatory postsynaptic potentials (fEPSPs) in layer II evoked by piriform cortex stimulation. An analysis of the receptor subtypes involved in the facilitation of evoked fEPSPs was conducted using horizontal slices of lateral entorhinal cortex in vitro. The effects of 15-min bath application of dopamine on synaptic responses were bidirectional and concentration dependent. Synaptic responses were enhanced by 10 microM dopamine and suppressed by concentrations of 50 and 100 microM. The D(1)-receptor antagonist SCH23390 (50 microM) blocked the significant facilitation of synaptic responses induced by 10 microM dopamine and the D(2)-receptor antagonist sulpiride (50 microM) prevented the suppression of fEPSPs observed with higher concentrations of dopamine. We propose here that dopamine release in the lateral entorhinal cortex, acting through D(1) receptors, can lead to an enhancement of the salience of sensory representations carried to this region from adjacent sensory cortices.     

Autoradiographic visualization of dopamine D-2 receptors in the monkey brain using the selective benzamide drug [3H]raclopride

Using the novel substituted benzamide drug [3H]raclopride in combination with in vitro receptor autoradiography, the distribution of dopamine D-2 receptors was studied in the monkey brain. Highest densities of D-2 receptors are present in dopamine-rich areas and the distribution shows the following rank order: caudatus and putamen greater than nucleus accumbens greater than olfactory tubercle greater than substantia nigra (pars compacta) greater than insular cortex greater than piriform and entorhinal cortex greater than substantia nigra (pars reticulata). In all of these areas [3H]raclopride binding was blocked by dopamine (1 microM) and by D-2 receptor antagonists such as (+)-butaclamol, eticlopride and raclopride, while the D-1 receptor antagonist SCH 23390 (1 microM) reduced [3H]raclopride binding by 15-20% in some restricted parts of the caudatus and putamen exclusively.     

The distribution and orientation of serotonin fibers in the entorhinal and other retrohippocampal areas. An immunohistochemical study with anti-serotonin antibodies in the rats brain

Summary The serotonin (5-hydroxytryptamine; 5-HT) innervation of the retrohippocampal region (subiculum, pre-and parasubiculum, area 29e, medial and lateral entorhinal area) in the rat brain has been examined with antibodies against 5-HT used in combination with fluorescence histochemistry. Analysis of consecutive sections cut in the coronal, sagittal, and horizontal planes revealed a widespread distribution of 5-HT immunoreactive fibers throughout the retrohippocampal region. This innervation was heterogeneous with regard to the morphological characteristics of the 5-HT fibers, their density and their spatial orientation.On the basis of morphological criteria, four different types of 5-HT positive processes were distinguished: (a) fine, convoluted fibers with small (0.5–0.8 m), round and evenly spaced varicosities; (b) fine fibers with elongated, irregularly distributed varicosities; (c) thick, possibly myelinated fibers, and (d) a terminal plexus with large (5–10 m), irregularly spaced varicosities. Analysis of the laminar distribution of the 5-HT fibers showed that whereas all layers contain 5-HT positive fibers, the molecular layer was the most densely innervated. The 5-HT fibers were found to be oriented both parallel and transverse to the longitudinal axis of medial and lateral entorhinal area. This grid-like arrangement was less pronounced in the presubiculum. Although the 5-HT innervation of the retrohippocampal region was found to be dominated by a widespread and apparently diffuse pattern, several areas contained dense clusters of preterminal 5-HT processes: area 29e, dorsal presubiculum (layer II), lateral entorhinal area (layer III and ventral layer II) and the transitional zone of the ventral entorhinal area. The 5-HT fibers were found to enter the retrohippocampal region primarily by three different routes; from the ventral and dorsal aspects and from the piriform and lateral neocortex (via the perirhinal area). Most of the fibers enter the region by the ventral route and these were found to ascend in all layers but predominantly in layer I.The location of the 5-HT cells giving rise to the innervation of the entorhinal area was studied by combining retrograde transport of fluorescent tracers with immunohistochemistry on the same tissue section. Both ipsi-and contralaterally located cells in the dorsal and median raphe nuclei were found to project to the entorhinal area. Most, but not all, of these retrogradely labeled cell bodies also contain 5-HT immunoreactivity.Abbreviations Used for Text and Figures ab angular bundle - ad area dentata - cc corpus callosum - dcp decussation of bracium conjunctivum - dr dorsal raphe nucleus (B7) - flm medial longitudinal fasciculus - mr median raphe nucleus (B8) - occ occipital cortex - rtp nucleus reticularis tegmentipontis - sub subiculum - vtG ventral tegmental area of Gudden - III third nerve nucleus - 28L entorhinal area, lateral part - 28L entorhinal area, lateral part (ventral) - 28M entorhinal area, medial part - 28M entorhinal area, medial part (ventral) - 49(a,b) parasubiculum - AMYG amygdala - Prs Presub-presubiculum - TA transitional area - TZ transitional zone     

Postnatal development of calcium-binding proteins immunoreactivity (parvalbumin, calbindin, calretinin) in the human entorhinal cortex

The entorhinal cortex is an essential component in the organization of the human hippocampal formation related to cortical activity. It transfers, neocortical information (ultimately distributed to the dentate gyrus and hippocampus) and receives most of the hippocampal output directed to neocortex. At birth, the human entorhinal cortex presents similar layer organization as in adults, although layer II (cell islands) and upper layer III have a protracted maturation. The presence of interneurons expressing calcium-binding proteins (parvalbumin, calbindin–D28K (calbindin) and calretinin) is well documented in the adult human entorhinal cortex. In many of them the calcium binding is co-localized with GABA. Parvalbumin-immunoreactive cells and fibers were virtually absent at birth, their presence increasing gradually in deep layer III, mostly in the lateral and caudal portions of the entorhinal cortex from the 5th month onwards. Calbindin immunoreactive cells and fibers were present at birth, mainly in layers II and upper III; mostly at rostral and lateral portions of the entorhinal cortex, increasing in number and extending to deep layers from the 5th month onwards. Calretinin immunoreactivity was present at birth, homogeneously distributed over layers I, II and upper V, throughout the entorhinal cortex. A substantial increase in the number of calretinin neurons in layer V was observed at the 5th month. The postnatal development of parvalbumin, calbindin and calretinin may have an important role in the functional maturation of the entorhinal cortex through the control of hippocampal, cortical and subcortical information.     

The time of origin of neurons in Ammon''s horn and the associated retrohippocampal fields

Summary The time of origin of the neurons in the hilus of the dentate gyrus, in the regio superior and regio inferior of Ammon's horn, and in the following retrohippocampal fields — the subiculum, presubiculum, parasubiculum, medial and lateral entorhinal areas and the perirhinal cortex, has been determined in the rat, by the technique of ³H-thymidine autoradiography. In each field the cells are generated over a limited period of about five days, with the majority being formed in a 24–48 h period. As in the neocortex, the cells in the various hippocampal fields are generated in a distinct inside-out sequence with respect to the ependymal zone in which they arise. In addition there are two distinct gradients along the transverse, or dentato-rhinal axis, of the formation. Thus the neurons in the hilar region of the dentate gyrus (including field CA₄) tend to arise earlier than the pyramidal cells in the regio inferior, and these in turn, are on average, generated earlier than those in the regio superior or in the subiculum. In the retrohippocampal region there is a comparable gradient extending medially from the perirhinal cortex to the presubiculum, the cells in the lateral entorhinal area being formed, on average, earlier than those in the medial entorhinal cortex, and these, in turn, are generated earlier than those in the para- or presubiculum. There is no evidence for a dorso-ventral (or septotemporal gradient) in any part of the hippocampal formation like that found in the dentate gyrus. Cell counts indicate that there are over 320,000 pyramidal cells in the regio superior (field CA₁) and about 150,000 in the regio inferior (fields CA₂ and CA₃).This work was supported in part by research grant NS 10943 from the National Institute of Neurological and Communicative Disorders and Stroke     

A projection from the deep layers of the entorhinal area to the hippocampal formation in the rat brain

The methods of retrograde fluorescent tracing and anterograde transport of the lectin Phaseolus vulgaris leucoagglutinin (PHA-L) were used to demonstrate the existence of projections from layers IV and VI of the entorhinal area to the hippocampal formation in the rat brain. These two layers of the medial and lateral entorhinal area innervate the molecular layer of Ammon's horn and the area dentata. In the area dentata the projection from layer IV follows that of the perforant path, while that from layer VI innervates the outer two-thirds of the molecular layer, the subgranular zone and the deep part of the hilus of the area dentata.