Cells In Stratum Oriens Research Articles

Glutamate released by Cajal-Retzius cells impacts specific hippocampal circuits and behaviors.

SUMMARYThe impact of Cajal-Retzius cells on the regulation of hippocampal circuits and related behaviors is unresolved. Here, we directly address this issue by impairing the glutamatergic output of Cajal-Retzius cells with the conditional ablation of vGluT2, which is their main vesicular glutamate transporter. Although two distinct conditional knockout lines do not reveal major alterations in hippocampal-layer organization and dendritic length of principal neurons or GABAergic cells, we find parallel deficits in specific hippocampal-dependent behaviors and in their putative underlying microcircuits. First, conditional knockout animals show increased innate anxiety and decreased feedforward GABAergic inhibition on dentate gyrus granule cells. Second, we observe impaired spatial memory processing, which is associated with decreased spine density and reduced AMPA/NMDA ratio of postsynaptic responses at the perforant- and entorhino-hippocampal pathways. We conclude that glutamate synaptically released by Cajal-Retzius cells is critical for the regulation of hippocampal microcircuits and specific types of behaviors.

Signaling properties of stratum oriens interneurons in the hippocampus of transgenic mice expressing EGFP in a subset of somatostatin‐containing cells

GABAergic interneurons constitute a heterogeneous group of cells that exert a powerful control on network excitability and are responsible for the oscillatory behavior crucial for information processing in the brain. These cells have been differently classified according to their morphological, neurochemical, and physiological characteristics. Here, whole cell patch clamp recordings were used to further characterize, in transgenic mice expressing EGFP in a subpopulation of GABAergic interneurons containing somatostatin (GIN mice), the functional properties of EGFP-positive cells in stratum oriens of the CA1 region of the hippocampus, in slice cultures obtained from P8 old animals. These cells showed passive and active membrane properties similar to those found in stratum oriens interneurons projecting to stratum lacunosum-moleculare. Moreover, they exhibited different firing patterns that were maintained upon membrane depolarization: irregular (48%), regular (30%), and clustered (22%). Trains of action potentials in interneurons evoked in a minority of principal cells (3/45) small amplitude GABAergic currents that at 20 Hz underwent short-term depression. In contrast, excitatory connections between principal cells and EGFP-positive interneurons were highly reliable (17/55) and exhibited a frequency and use-dependent facilitation particularly in the gamma band. In addition, recordings from paired of interconnected EGFP-positive cells revealed in 47% of the cases electrical coupling, which was abolished by carbenoxolone (200 microM). On average, the coupling coefficient was 0.21 +/- 0.07. When electrical coupling was particularly strong it acted as a powerful low-pass filter, thus contributing to alter the output of individual cells. In conclusion, it appears that the dynamic interaction between cells with various firing patterns could differently affect GABAergic signaling, leading, as suggested by simulation data, to a wide range of interneuronal communication within the hippocampal network.

Populations of hippocampal inhibitory neurons express different levels of cytochrome c

Cytochrome c (CC) immunoreactivity was quantified in functionally distinct rat hippocampal inhibitory neuron populations using double immunocytochemistry and laser scanning confocal microscopy to measure the CC expression level as well as the amount of mitochondria within the cells, which is a sign of neuronal activity. The CC signal showed a similar distribution to cytochrome c oxidase histochemical staining. Strongly stained somata, dendrites and axon terminal clouds were dispersed over the low intensity neuropil staining. The staining was granular and electron microscopic investigation confirmed that the signal was localized in mitochondria. Intensively labeled neurons, showing the morphological features of inhibitory cells, were most frequently found in the principal cell layers, stratum oriens of the CA1-3 areas, stratum lucidum and hilus. These neurons contained not only a higher number of mitochondria than the principal cells but the intensity of the mitochondrial staining was evidently stronger. Among the examined interneuronal populations, parvalbumin-immunoreactive neurons were intensively labeled for CC. Calbindin D28k- (CB), somatostatin- and cholecystokinin-labeled cells showed heterogeneous CC levels, whereas calretinin-immunoreactive cells never showed a strong CC signal. CB cells in stratum oriens and alveus layers, lucidum and the hilus were strongly labeled for CC. CB cells in such regions are known to project to the medial septum and contain somatostatin. We have demonstrated that the CA1 interneurons that project to the medial septum (hippocampo-septal neurons) express a high level of CC. Thus, similar to the parvalbumin-containing basket and axo-axonic cells, the hippocampo-septal neurons potentially have a high average activity level.

Excitatory inputs to CA1 interneurons show selective synaptic dynamics.

The dynamic properties of synapses between neurons in the hippocampal CA1 area are important for the frequency-dependent signal transfer of the network. We have examined the synaptic dynamics of excitatory inputs to CA1 interneurons and pyramidal cells using whole cell voltage-clamp recordings. The CA1 network was activated using extracellular stimulation electrodes at the Schaffer collaterals (feedforward activation) or at the Alveus (activation of the feedback loop). The dynamic properties of input from the Schaffer collaterals to CA1 interneurons (basket and bistratified cells) were different from the synaptic dynamics of input from the Alveus. Synaptic input from the Schaffer collaterals to CA1 interneurons showed facilitation for most frequencies. After 10 stimuli the synaptic response reached a plateau level that was approximately 150% of the first response in the train. In contrast, the plateau levels of Alveus inputs to interneurons were not different from the first responses for frequencies <or=40 Hz. Paired-pulse facilitation of Schaffer input was stronger than for Alveus input. Cells in stratum oriens with horizontal dendritic trees appeared to be a special group of interneurons because Alveus input to these cells showed strong facilitation with plateau levels of 200% of the first responses. Schaffer input to CA1 basket and bistratified cells showed similar synaptic dynamics compared with Schaffer input to pyramidal cells for frequencies <or=80 Hz. The synaptic dynamics of Schaffer and Alveus input depended only weakly on the stimulus intensity. The difference between the dynamics of Alveus and Schaffer input to CA1 interneurons implies that the relative contribution of feedforward and -back inhibition to network activity depends on the frequency of the input signal at the afferent fibers, adding a level of complexity to transient responses.

Developmental changes in calpain activity, GluR1 receptors and in the effect of kainic acid treatment in rat brain

The cellular distribution of calpain activation and glutamate receptor 1 (GluR1) subunits of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and their alterations following kainic acid-induced seizure were evaluated during postnatal development using antibodies specific for spectrin breakdown product and the C-terminus of GluR1 subunits. In the first postnatal week, most brain regions exhibited high levels of calpain activity that progressively decreased during the following weeks. The highest levels of spectrin breakdown product immunoreactivity were observed in the somata and proximal dendrites of hippocampal pyramidal cells, non-pyramidal neurons in stratum oriens, and cortical neurons. In general, during the first two postnatal weeks, kainic acid treatment induced a decrease in spectrin breakdown product immunoreactivity in neuronal cell bodies and an increase in dendritic fields. Obvious elevation in spectrin breakdown product immunoreactivity in selective non-pyramidal cells in stratum oriens started at postnatal day 14, and was further evidenced by postnatal day 21. Likewise, massive calpain activation in subpopulations of neurons in some thalamic nuclei, amygdala, and pyriform cortex was observed after the third postnatal week. GluR1 subunits were highly expressed throughout the forebrain in the first postnatal week, further increased during the second postnatal week, decreased thereafter, and reached adult levels after postnatal day 21. In cortex, intense GluR1 immunostaining was found in the somata and proximal processes of pyramidal and non-pyramidal neurons, with the non-pyramidal neurons in layers IV through VI exhibiting the densest immunolabelling. In the first two postnatal weeks, the somata of hippocampal pyramidal neurons exhibited intense GluR1 immunostaining that became more dendritic in the subsequent developmental period. While hilar cells exhibited a similar developmental pattern as CA regions, the molecular layer of dentate gyrus exhibited weak immunoreactivity from postnatal day 7 to postnatal day 14. The early increase in GluR1 immunoreactivity in hippocampal pyramidal layer following kainic acid treatment occurred throughout the developmental period, while the later decrease in CA regions, amygdala, and pyriform cortex was observed only in postnatal day 21 animals. The combined immunocytochemical studies of spectrin breakdown product localization and GluR1 expression indicate that calpain activation might play an important role in synaptic formation, developmental regulation of synaptic plasticity, and neuronal vulnerability to excitotoxicity during postnatal development. Moreover, calpain-mediated modulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors might underlie these processes.

Distinct interneuron types express m2 muscarinic receptor immunoreactivity on their dendrites or axon terminals in the hippocampus

In previous studies m2 muscarinic acetylcholine receptor-immunoreactive interneurons and various types of m2-positive axon terminals have been described in the hippocampal formation. [43]The aim of the present study was to identify the types of interneurons expressing m2 receptor and to examine whether the somadendritic and axonal m2 immunostaining labels the same or distinct cell populations. In the CA1 subfield, neurons immunoreactive for m2 have horizontal dendrites, they are located at the stratum oriens/alveus border and have an axon that project to the dendritic region of pyramidal cells. In the CA3 subfield and the hilus, m2-positive neurons are multipolar and are scattered in all layers except stratum lacunosum-moleculare. In stratum pyramidale of the CA1 and CA3 regions, striking axon terminal staining for m2 was observed, surrounding the somata and axon initial segments of pyramidal cells in a basket-like manner. The co-localization of m2 with neurochemical markers and GABA was studied using the “mirror” technique and fluorescent double-immunostaining at the light microscopic level and with double-labelling using colloidal gold-conjugated antisera and immunoperoxidase reaction (diaminobenzidine) at the electron microscopic level. GABA was shown to be present in the somata of most m2-immunoreactive interneurons, as well as in the majority of m2-positive terminals in all layers. The calcium-binding protein parvalbumin was absent from practically all m2-immunoreactive cell bodies and dendrites. In contrast, many of the terminals synapsing on pyramidal cell somata and axon initial segments co-localized parvalbumin and m2, suggesting a differential distribution of m2 receptor immunoreactivity on the axonal and somadendritic membrane of parvalbumin-containing basket and axo-axonic cells. The co-existence of m2 receptors with the calcium-binding protein calbindin and the neuropeptides cholecystokinin and vasoactive intestinal polypeptide was rare throughout the hippocampal formation. Only calretinin and somatostatin showed an appreciable degree of co-localization with m2 (20% and 15%, respectively). Using retrograde tracing, some of the m2-positive cells in stratum oriens were shown to project to the medial septum, accouting for 38% of all projection neurons. The present results demonstrate that there is a differential distribution of m2 receptor immunoreactivity on the axonal vs the somadendritic membranes of distinct interneuron types and suggest that acetylcholine via m2 receptors may reduce GABA release presynaptically from the terminals of perisomatic inhibitory cells, while it may act to increase the activity of another class of interneuron, which innervates the dendritic region of pyramidal cells.

Axonal sprouting of CA1 pyramidal cells in hyperexcitable hippocampal slices of kainate-treated rats.

CA1 pyramidal cells become hyperexcitable following hippocampal kainate lesions. To examine if axonal sprouting contributes to this epileptiform activity, the local axonal arborization of CA1 pyramidal cells was examined after intracellular labelling with biocytin in hippocampal slices from control rats and in hyperexcitable slices obtained from rats treated with kainate (bilateral intracerebroventricular injections) 2-4 weeks previously. Biocytin-labelled cells with an axon that could be followed from the soma to the alveus were drawn and reconstructed with a camera lucida (15 cells from control slices and 14 cells from hyperexcitable slices). Local axonal arborizations were more extensive in cells of hyperexcitable slices. This increase in axon collaterals was generally seen in the alveus and in stratum oriens, but changes were more prominent in the latter. In stratum oriens, cells from hyperexcitable slices showed a significant increase in mean total axon length (1035 versus 373 mu m in control), in mean number of branching points (6.50 versus 0.67 in control) and in mean number of segment orders per axon (3.07 versus 1.47 in control). Their first-order axon segments were similar in length to those of control cells (236 versus 338 pm in control), but with significantly more branching points (2.86 versus 0.53 in control). Their second-order axon segments were significantly longer (381 versus 63 mu m in control) and also showed more branching points (2.71 versus 0.13 in control). Their third- and fourth-order axon segments were also longer and with more branching points. Under high-power light microscopic examination, biocytin-labelled axonal varicosities in cells of hyperexcitable slices were often seen in close apposition with their own dendrites, presumably making synaptic contact (five of nine cells examined). No such appositions were seen in any of the control cells (seven cells examined). These results indicate that, following kainate lesions, there is sprouting of local axon collaterals of CA1 pyramidal cells in stratum oriens and in the alveus. This local increase in axon collaterals may contribute to the epileptiform activity in the CA1 area by providing recurrent excitation via newly formed synaptic, and perhaps even autaptic, contacts with pyramidal cell dendrites.

Calbindin D 28k-containing nonpyramidal cells in the rat hippocampus: Their immunoreactivity for GABA and projection to the medial septum

Calbindin D 28k-containing non-pyramidal cells were found in all layers and subfields of the hippocampus, with the highest frequency in stratum radiatum of the CA1–CA3 subfields. A large number of these neurons had a vertically oriented dendritic tree, often restricted to stratum radiatum. In stratum oriens and near to the border of strata radiatum and lacunosum moleculare cells with horizontally running dendrites were also found. Multipolar cells were most common in stratum radiatum of the CA3 region. The GABAergic nature of the calbindin D 28k-containing non-pyramidal cells was studied using the “mirror” technique. Adjacent thick sections were immunostained for calbindin D 28k and GABA, and halved neurons were identified on the common surfaces. The majority of calbindin D 28k-containing non-pyramidal cells were shown to be GABAergic. The GABA-negative calbindin cells were found in relatively large numbers in stratum oriens of the CA1–CA3 region, and occasionally in strata radiatum and pyramidale of CA2, and in stratum radiatum of the CA3c region near to the border of the dentate hilus. However, even in these cells a weak immunostaining, only slightly but consistently above background level, was always observed. Earlier studies have demonstrated that the somata of GABAergic neurons with distant projections may contain a level of GABA that is below the detection threshold of immunocytochemistry. Here we provide direct evidence that the calbindin-containing non-pyramidal cells were among those projecting to the medial septum. Following horseradish peroxidase injections into the medial septum 80% of the retrogradely labelled non-pyramidal cells were found to be immunoreactive for calbindin D 28k, and 20% contained neuropeptide Y. These results suggest that the calbindin D 28k-containing and apparently GABA-immunonegative non-pyramidal cells in stratum oriens of the CAl–CA3 regions may also be GABAergic, but have a distant projection, that is, to the medial septum.

Innervation of different peptide-containing neurons in the hippocampus by gabaergic septal afferents

The termination pattern of septohippocampal axons visualized by anterograde transport of Phaseolus vulgaris leucoagglutinin was studied in the hippocampal formation in the rat, with special reference to the innervation of neurons immunoreactive for the neuroactive peptides cholecystokinin, somatostatin or vasoactive intestinal polypeptide. The type I, GABAergic, septohippocampal afferents were shown to terminate on neurons immunoreactive for each of the three peptides. The cholecystokinin-like immunoreactive neurons in all regions, and the somatostatin-immunoreactive cells in stratum oriens of CA1 region were the most preferred targets. Cholecystokinin-immunoreactive cells, especially those in the granule cell layer of the dentate gyrus, were often seen to be contacted by type II (presumed cholinergic) axons as well. The somatostatin-immunoreactive cells in the hilus were also innervated by type I septohippocampal axons, although less frequently than those in stratum oriens of the CA1 subfield. Each type of peptidergic neuron received multiple symmetrical synaptic input from the Phaseolus vulgaris leucoagglutinin-labelled septal afferents, as confirmed by correlated electron microscopy. The majority of these neuropeptide-containing cells are known to be GABAergic, and to have distinct input and output relationships. Thus, the present results demonstrate that the GABAergic septohippocampal pathway can control a wide range of putative inhibitory circuits, and thereby influence the pattern of electrical activity in the hippocampal formation.

Fine structure of identified neurons in the primate hippocampus: A combined Golgi/EM study in the baboon

The hippocampi of two 1-year-old female baboons (Papio anubis) were used for a combined Golgi/electron microscope (EM) study of characteristic cell types in the hippocampus proper and fascia dentata. Results were compared with previous Golgi/EM studies of hippocampal neurons in small laboratory animals. Cell bodies of pyramidal neurons in CA1 were more loosely distributed than known from studies on the rat or guinea pig. Numerous basal and horizontal dendrites originating from the perikaryon filled in the space between neighboring cell bodies. Apical stem dendrites were varying in length, depending on the position of the parent cell body in outer or inner portions of the pyramidal layer. Dendrites were densely covered with spines which in the EM showed very complex synaptic contacts. In contrast to our observations in rats and guinea pigs, CA3 pyramidal cells in the monkey hippocampus exhibited numerous large spines or excrescences not only on apical dendrites but also on basal dendrites running through stratum oriens. These excrescences appeared to be more complex than in small rodents. They often branched, protruding deeply into presynaptic mossy fiber boutons, and formed multiple asymmetric synaptic contacts. Granule cells of the monkey fascia dentata, in contrast to those of the rodent, occasionally had basal dendrites extending into the hilar region. In the EM, granule cells either with or without basal dendrites exhibited fine structural characteristics that were very similar to those described in Golgi/EM studies of granule cells in the rat fascia dentata. Of the various types of nonpyramidal neurons the horizontal cells in stratum oriens with dendrites parallel to the alveus were analyzed. As seen in rats, these cells exhibited large amounts of rough endoplasmic reticulum, indentations of the nuclear membrane, and nuclear inclusions. Numerous terminals formed synaptic contacts on dendritic shafts. In contrast to rodents, numerous spines arose from dendrites and cell bodies of these neurons. In the EM, often single spines were found to establish synaptic contacts with several presynaptic boutons. In summary, our correlated light and EM study of four characteristic cell types, which are present in both nonprimates and primates, demonstrates a much more complex dendritic pattern and synaptic organization of these neurons in primates than in commonly studied small laboratory animals.

Various types of non-pyramidal hippocampal neurons project to the septum and contralateral hippocampus

The morphology was studied of hippocampal neurons which had their somata in the hilus of the area dentata, and in stratum radiatum or stratum oriens of Ammon's horn, and which sent projections to the septum and contralateral hippocampus, respectively. The fluorescent marker Fast Blue was injected into the septum or contralateral hippocampus. Somata were then identified by their fluorescent label in slices of perfused brains. After intracellular injection of these somata with Lucifer Yellow, it was found that contralaterally projecting neurons were pyramidal cells, inverted fusiform and multipolar cells in CA3c, and stellate, fusiform and multipolar cells in the hilus. After septal injections, we identified two groups of aspiny stellate cells in the hilus; pyramidal basket cells, polygonal basket cells, horizontal basket cells in stratum oriens; and stellate cells in stratum radiatum of CA1 and CA3, as well as pyramid-like aspiny cells in stratum radiatum of CA1. These cells also had short locally arborizing axons, thus probably contributing to local circuits. Such cells may constitute a third class of hippocampal neurons combining the properties of principal cells and interneurons. These results support the opinion that the simple concept of separating hippocampal cells into projection neurons and local-circuit neurons needs reconsideration.