Pyramidal-like Cells Research Articles

Morfologi Kelenjar Parotis Dan Mandibularis Babi Timor (Sus scrofa domesticus)

Timor pig (Sus scrofa domesticus) is classified as one of local Indonesia pigs breed of Sus scrofa. Salivary glands had huge contribution to the digestive system. This research aimed to identify the gross anatomy and histomorphology of the parotid and mandibular glands in Timor pigs. Five samples were taken from Atambua slaughter house. Observation of gross anatomy were carried out directed in slaughter house. Then, the glands were fixated in 10% formalin and stained with HE. The results showed the parotid gland was located on the ventral of the auricle and stretched superficially along the caudal masseter muscle to the caudal of the lower jaw. The parotid gland had brown triangle-shape lobules. The mandibular gland was located in the caudal of the lower jaw or profundus of the parotid gland and had pinkish irregular-shape with rounded edges. The asinar cells of the parotid gland were dominated by serous acini with pyramid-like cells. Serous cells were round-shaped in center of cell membrane. Other serous cells called special serous cells were round-shaped pressed into basal of cell membrane. The asinar cells of the mandibular gland were dominated by mucous acini and discovered serous acini and serous demilune. Pyramid-like mucous cells had flat nucleus in basal of cell membrane. Pyramid-like serous cells had round nucleus in center of cell membrane. Serous cell that arranged in peripheral mucous acini will form into serous demilune. The ducts on the parotid and mandibular glands comprised of the intercalated ducts, the striated ducts and the excretory ducts.

Cortical circuit activity underlying sleep slow oscillations and spindles

Slow oscillations and sleep spindles are hallmarks of the EEG during slow-wave sleep (SWS). Both oscillatory events, especially when co-occurring in the constellation of spindles nesting in the slow oscillation upstate, are considered to support memory formation and underlying synaptic plasticity. The regulatory mechanisms of this function at the circuit level are poorly understood. Here, using two-photon imaging in mice, we relate EEG-recorded slow oscillations and spindles to calcium signals recorded from the soma of cortical putative pyramidal-like (Pyr) cells and neighboring parvalbumin-positive interneurons (PV-Ins) or somatostatin-positive interneurons (SOM-Ins). Pyr calcium activity was increased more than threefold when spindles co-occurred with slow oscillation upstates compared with slow oscillations or spindles occurring in isolation. Independent of whether or not a spindle was nested in the slow oscillation upstate, the slow oscillation downstate was preceded by enhanced calcium signal in SOM-Ins that vanished during the upstate, whereas spindles were associated with strongly increased PV-In calcium activity. Additional wide-field calcium imaging of Pyr cells confirmed the enhanced calcium activity and its widespread topography associated with spindles nested in slow oscillation upstates. In conclusion, when spindles are nested in slow oscillation upstates, maximum Pyr activity appears to concur with strong perisomatic inhibition of Pyr cells via PV-Ins and low dendritic inhibition via SOM-Ins (i.e., conditions that might optimize synaptic plasticity within local cortical circuits).

Two distinct types of depolarizing afterpotentials are differentially expressed in stellate and pyramidal-like neurons of entorhinal-cortex layer II.

Two types of principal neurons, stellate cells and pyramidal-like cells, are found in medial entorhinal-cortex (mEC) layer II, and are believed to represent two distinct channels of information processing and transmission in the entorhinal cortex-hippocampus network. In this study, we found that depolarizing afterpotentials (DAPs) that follow single action potentials (APs) evoked from various levels of holding membrane voltage (Vh ) show distinct properties in the two cells types. In both, an evident DAP followed the AP at near-threshold Vh levels, and was accompanied by an enhancement of excitability and spike-timing precision. This DAP was sensitive to voltage-gated Na(+)-channel block with TTx, but not to partial removal of extracellular Ca(2+). Application of 5-μM anandamide, which inhibited the resurgent and persistent Na(+) -current components in a relatively selective way, significantly reduced the amplitude of this particular DAP while exerting poor effects on the foregoing AP. In the presence of background hyperpolarization, DAPs showed an opposite behavior in the two cell types, as in stellate cells they became even more prominent, whereas in pyramidal-like cells their amplitude was markedly reduced. The DAP observed in stellate cells under this condition was strongly inhibited by partial extracellular-Ca(2+) removal, and was sensitive to the low-voltage-activated Ca(2+)-channel blocker, NNC55-0396. This Ca(2+) dependence was not observed in the residual DAP evoked in pyramidal-like cells from likewise negative Vh levels. These results demonstrate that two distinct mechanism of DAP generation operate in mEC layer-II neurons, one Na(+)-dependent and active at near-threshold Vh levels in both stellate and-pyramidal-like cells, the other Ca(2+)-dependent and only expressed by stellate cells in the presence of background membrane hyperpolarization.

Diversity of Reporter Expression Patterns in Transgenic Mouse Lines Targeting Corticotropin-Releasing Hormone-Expressing Neurons.

Transgenic mice, including lines targeting corticotropin-releasing factor (CRF or CRH), have been extensively employed to study stress neurobiology. These powerful tools are poised to revolutionize our understanding of the localization and connectivity of CRH-expressing neurons, and the crucial roles of CRH in normal and pathological conditions. Accurate interpretation of studies using cell type-specific transgenic mice vitally depends on congruence between expression of the endogenous peptide and reporter. If reporter expression does not faithfully reproduce native gene expression, then effects of manipulating unintentionally targeted cells may be misattributed. Here, we studied CRH and reporter expression patterns in 3 adult transgenic mice: Crh-IRES-Cre;Ai14 (tdTomato mouse), Crfp3.0CreGFP, and Crh-GFP BAC. We employed the CRH antiserum generated by Vale after validating its specificity using CRH-null mice. We focused the analyses on stress-salient regions, including hypothalamus, amygdala, bed nucleus of the stria terminalis, and hippocampus. Expression patterns of endogenous CRH were consistent among wild-type and transgenic mice. In tdTomato mice, most CRH-expressing neurons coexpressed the reporter, yet the reporter identified a few non-CRH-expressing pyramidal-like cells in hippocampal CA1 and CA3. In Crfp3.0CreGFP mice, coexpression of CRH and the reporter was found in central amygdala and, less commonly, in other evaluated regions. In Crh-GFP BAC mice, the large majority of neurons expressed either CRH or reporter, with little overlap. These data highlight significant diversity in concordant expression of reporter and endogenous CRH among 3 available transgenic mice. These findings should be instrumental in interpreting important scientific findings emerging from the use of these potent neurobiological tools.

Cytological organization of the alpha component of the anterior olfactory nucleus and olfactory limbus

This study describes the microscopic organization of a wedge-shaped area at the intersection of the main (MOB) and accessory olfactory bulbs (AOBs), or olfactory limbus (OL), and an additional component of the anterior olfactory nucleus or alpha AON that lies underneath of the AOB. The OL consists of a modified bulbar cortex bounded anteriorly by the MOB and posteriorly by the AOB. In Nissl-stained specimens the OL differs from the MOB by a progressive, antero-posterior decrease in thickness or absence of the external plexiform, mitral/tufted cell, and granule cell layers. On cytoarchitectual grounds the OL is divided from rostral to caudal into three distinct components: a stripe of glomerular-free cortex or preolfactory area (PA), a second or necklace glomerular area, and a wedge-shaped or interstitial area (INA) crowned by the so-called modified glomeruli that appear to belong to the anterior AOB. The strategic location and interactions with the main and AOBs, together with the previously noted functional and connectional evidence, suggest that the OL may be related to both sensory modalities. The alpha component of the anterior olfactory nucleus, a slender cellular cluster (i.e., 650 × 150 μm) paralleling the base of the AOB, contains two neuron types: a pyramidal-like neuron and an interneuron. Dendrites of pyramidal-like cells (P-L) organize into a single bundle that ascends avoiding the AOB to resolve in a trigone bounded by the edge of the OL, the AOB and the dorsal part of the anterior olfactory nucleus. Utrastructurally, the neuropil of the alpha component contains three types of synaptic terminals; one of them immunoreactive to the enzyme glutamate decarboxylase, isoform 67.

Distinctive hippocampal CA2 subfield of the Amazon rodent Proechimys

Previous data of our laboratory have shown that the Amazonian rodents Proechimys do not present spontaneous seizures in different models of epilepsy, suggesting endogenous inhibitory mechanisms. Here, we describe a remarkably different Proechimy's cytoarchitecture organization of the hippocampal cornu Ammonis 2 (CA2) subfield. We identified a very distinctive Proechimy′s CA2 sector exhibiting disorganized cell presentation of the pyramidal layer and atypical dispersion of the pyramidal-like cells to the stratum oriens, strongly contrasting to the densely packed CA2 cells in the Wistar rats. Studies showed that CA2 is the only cornu ammonis (CA) subfield resistant to the extensive pyramidal neural loss in mesial temporal lobe epilepsy (MTLE) associated to hippocampal sclerosis. Thus, in order to investigate this region, we used Nissl and Timm staining, stereological approach to count neurons and immunohistochemistry to neuronal nuclei (NeuN), parvalbumin (PV), calbindin (CB) and calretinin (CR). We did not notice statistically significant differences in the total number of neurons of the CA2 region between Proechimys and Wistar. However, Proechimys rodents presented higher CA2 volume than Wistar rats. Furthermore, no significant difference in the optical density of parvalbumin-immunoreactivity was found between subject groups. On the other hand, Proechimys presented significant higher density of calbindin and calretinin-immunoreactivity when compared to Wistar rats. In this context, this unique CA2 subfield seen in Proechimys opens up a new set of possibilities to explore the contribution of CA2 neurons in normal and pathological brain circuits.

Interaction of dopamine D1 with NMDA NR1 receptors in rat prefrontal cortex

Despite the tremendous importance of D1 and NMDA receptors to cognition (working memory, executive functions) and synaptic plasticity in the prefrontal cortex (PFC), little is known about the molecular mechanisms underlying D1–NMDA receptors interactions in this brain area. Here, we show that D1 receptors and the NMDA receptor co-localize in single pyramidal neurons and interneurons in adult rat PFC. NR1 and NR2A expression are found in different neuronal types. Conversely, D1 receptors are predominantly localized in pyramidal-like cells and parvalbumin positive cells. NR1 co-immunoprecipitates with D1 receptor in adult medial PFC. In prefrontal primary cultures, NMDA does not affect the D1 receptor dependent-cAMP production. In contrast, activation of D1 receptor potentiates the NMDA mediated increase in cytosolic Ca 2+, an effect that was blocked by a PKA inhibitor. We conclude that D1 receptor potentiates the NMDA-Ca 2+ signal by a PKA-dependent mechanism.

Ancestry of the Mammalian Preplate and its Derivatives: Evolutionary Relicts or Embryonic Adaptations?

Mammalian cortical development is preceded by the elaboration of a transient preplate, which is split into a superficial marginal zone and a deep subplate after the arrival of the cortical plate. There has been some controversy in the evolutionary interpretation of this transient structure, as some propose it to represent the ancestral cortex or pallium of non-mammals, while others consider it to be a phylogenetic novelty. The preplate and its derivatives contain components derived by both tangential and radial migration. Tangentially migrating elements include pioneer neurons and interneurons, both of subpallial origin, and Cajal-Retzius cells, mostly of pallial origin. Pioneer neurons were probably present in the ancestors of mammals, but may have changed their original superficial position to one below the developing cortex, thus attracting thalamic afferents in the subcortical white matter, and making them penetrate the cortex radially. In mammals, Cajal-Retzius cells appear to have increased both in number and on their level of reelin expression, perhaps in the context of controlling the final stages of migration in a radially expanding neoocortex. Radial-migrating cells are partly represented by the pyramidal-like cells of the subplate. These neurons resemble the excitatory elements of the adult reptilian cortex, but is not clear whether they are their true homologues. One possibility is that these cells appeared by virtue of a heterochronic process in which the earliest radial elements of the cortical plate began to be produced at progressively earlier developmental stages. Thus, we conclude that the mammalian preplate and its derivatives contain both ancestral and derived elements, all of which have been modified in the course of mammalian evolution to support an increasingly complex cortical plate development.

Abnormal Cortical Cells and Astrocytomas in the Eker Rat Model of Tuberous Sclerosis Complex

In patients with tuberous sclerosis complex (TSC), a wide range of neurologic abnormalities develop, including mental retardation and seizures. Brains from TSC patients are characterized by the presence of cortical tubers, large dysmorphic neurons, and abnormal cytomegalic cells. Although analysis of human TSC brain samples led to the identification of these abnormal cell types, very little is known about how these cells function. In an effort to model TSC-associated CNS abnormalities (and ultimately to analyze the electrophysiologic properties of abnormal cells), we examined Eker rats carrying a Tsc2 mutation. Anatomic studies, including standard histologic stains and immunocytochemistry, were performed on young Eker rats exposed to a carcinogen in utero or aged untreated Eker rats (18-24 months old). Pregnant TSC2+/- females were injected once a day with hydroquinone (HQ), and offspring were killed at postnatal day P14 or P28. Coronal tissue sections throughout the CNS were prepared and stained for cresyl violet. In separate studies, brains of old untreated Eker rats were sectioned for anatomic analysis by using standard immunohistochemical techniques. Tissue sections stained with cresyl violet did not reveal any gross differences between HQ-treated Eker (Tsc2Ek/+) rats and siblings (Tsc2+/+). However, two classes of abnormal giant cells were observed in brain sections from untreated aged Eker rats: (a) large dysmorphic pyramid-like cells immunoreactive for NeuN, tuberin, and EAAC-1 in layers IV-VI; and (b) abnormal cytomegalic cells immunoreactive for glial fibrillary acidic protein (GFAP), vimentin, and nestin in deep cortical layers or along the white matter. In addition, large subependymal astrocytomas were observed in four animals. Our data suggest that cortical tuber formation in Eker rats is a rare event and that prenatal exposure to a nongenotoxic carcinogen such as HQ is not sufficient to induce tuber formation. However, with advanced age, an increased likelihood of astrocytoma formation and the emergence of dysmorphic neurons and cytomegalic cells in the Eker rat brain might exist; each of these abnormalities mimics those seen clinically and could contribute to neurologic problems associated with TSC. Further analysis of this rodent model may be warranted.

γ-Protocadherins Are Targeted to Subsets of Synapses and Intracellular Organelles in Neurons

The clustered protocadherins (Pcdhs) comprise >50 putative synaptic recognition molecules that are related to classical cadherins and highly expressed in the nervous system. Pcdhs are organized into three gene clusters (alpha, beta, and gamma). Within the alpha and gamma clusters, three exons encode the cytoplasmic domain for each Pcdh, making these domains identical within a cluster. Using an antibody to the Pcdh-gamma constant cytoplasmic domain, we find that all interneurons in cultured hippocampal neurons express high levels of Pcdh-gamma(s) in a nonsynaptic distribution. In contrast, only 48% of pyramidal-like cells expressed appreciable levels of these molecules. In these cells, Pcdh-gamma(s) were associated with a subset of excitatory synapses in which they may mediate presynaptic to postsynaptic recognition in concert with classical cadherins. Immunogold localization in hippocampal tissue showed Pcdh-gamma(s) at some synapses, in nonsynaptic plasma membranes, and in axonal and dendritic tubulovesicular structures, indicating that they may be exchanged among synapses and intracellular compartments. Our results show that although Pcdh-gamma(s) can be synaptic molecules, synapses form lacking Pcdh-gamma(s). Thus, Pcdh-gamma(s) and their relatives may be late additions to the classical cadherin-based synaptic adhesive scaffold; their presence in intracellular compartments suggests a role in modifying synaptic physiology or stability.

Differentiation of the RN33B Cell Line into Forebrain Projection Neurons after Transplantation into the Neonatal Rat Brain

The rat neural cell line RN33B has a remarkable ability to undergo region-specific neuronal differentiation after transplantation into the CNS. To further study its neurogenic properties in vivo, we used a recombinant lentiviral vector to genetically label the cells with the Green Fluorescent Protein (GFP) gene before implantation into the striatum/cortex, hippocampus, or mesencephalon of newborn rats. Three weeks after implantation, about 1–2% of the GFP-expressing cells had developed morphologies typical of neurons, astrocytes, or oligodendrocytes, the rest remained as either immature or undifferentiated nestin-positive cells. At 15–17 weeks postgrafting, the immature cells had disappeared in most graft recipients and only cells with neuronal or glial morphologies remained in similar numbers as at 3 weeks. The GFP distributed throughout the expressing cells, revealing fine morphological details, including dendrites with spines and extensive axonal projections. In all forebrain regions, the grafted cells differentiated into neurons with morphologies characteristic for each site, including large numbers of pyramidal-like cells in the cortex and the hippocampus, giving rise to dense projections to normal cortical target regions and to the contralateral hippocampus, respectively. In lower numbers, it was also possible to identify GFP-positive granulelike cells in the hippocampus, as well as densely spiny neurons in the striatum. In the mesencephalon by contrast, cells with astrocytic features predominated. The ability of the grafted RN33B cells to undergo region-specific differentiation into highly specialized types of forebrain projection neurons and establish connections with appropriate targets suggests that cues present in the microenvironment of the neonatal rat brain can effectively guide the development of immature progenitors, also in the absence of ongoing neurogenesis.

Expression changes of somatostatin receptor subtypes sst2A, sst2B, sst3 and sst4 after a cortical contusion trauma in rats

The neuropeptide somatostatin acts as a neuromodulator in the CNS in a predominantly inhibitory manner. In this study, an ipsilateral cortical and hippocampal damage in the brain of adult rats was induced by a cortical contusion trauma in order to examine subsequent changes of expression of different somatostatin receptor subtypes (sst). By using subtype specific antibodies we found a clear decline of expression level for sst2A, sst2B, sst3 and sst4 subtypes in the pyramidal cell layer of the ipsilateral hippocampus. Nissl staining revealed that this decline of expression level is due to cell death of sst expressing neurons within the first 48 h after trauma. Additionally we found a progressive infiltration of sst4 positive cells into regions of cortical and hippocampal damage. The number of these cells increases strikingly within the first 3 days after trauma and it seems that their morphology changes from a round to an astrocyte-like shape. Moreover, sst4 and sst2A positive cells accumulate in the ipsilateral ependym and pyramidal-like cells expressing sst4 were found beneath the damaged CA3 pyramidal layer. Taken together, after trauma we found deterioration of sst positive neurons and an additional activation of sst4 and sst2A expressing cells the final fate of which has to be elucidated further.

Intact sorting, targeting, and clustering of gamma-aminobutyric acid A receptor subtypes in hippocampal neurons in vitro.

The cellular and subcellular distribution of four GABA(A) receptor subtypes, identified by the presence of the alpha1, alpha2, alpha3, or alpha5 subunit, was investigated immunocytochemically in dissociated cultures of hippocampal neurons. We addressed the questions whether (1) cell-type specific expression, (2) axonal/somatodendritic targeting, and (3) synaptic/extrasynaptic clustering of GABA(A) receptor subtypes was retained in vitro. For comparison, the in vivo distribution pattern was assessed in sections from adult rat brain. The differential expression of GABA(A) receptor subunits allowed to identify five morphologically distinct cell types in culture: the alpha1 subunit was observed in glutamic acid decarboxylase-positive interneurons, the alpha2 and alpha5 subunits marked pyramidal-like cells, and the alpha3 subunit labeled three additional cell types, including presumptive hilar cells. All subunits were found in the somatodendritic compartment. In addition, appropriate axonal targeting was evidenced by the intense alpha2, and sometimes alpha3 subunit labeling of axon-initial segments (AIS) of pyramidal cells and hilar cells, respectively. Accordingly, both receptor subtypes were targeted to AIS in vivo, as well. Synaptic receptors were identified by colocalization with gephyrin, a postsynaptic clustering protein, and apposition to presynaptic terminals labeled with synapsin I. In vitro and in vivo, alpha1- and alpha2-receptor subtypes formed numerous synaptic clusters, alpha3-GABA(A) receptors were located either synaptically or extrasynaptically depending on the cell type, whereas alpha5-GABA(A) receptors were extrasynaptic. We conclude that receptor targeting to broad subcellular locations does not require specific GABAergic innervation patterns, which are disturbed in vitro, but depends on protein-protein interactions in the postsynaptic cell that are both subunit- and neuron-specific.

Primary CA1 and conditionally immortal MHP36 cell grafts restore conditional discrimination learning and recall in marmosets after excitotoxic lesions of the hippocampal CA1 field.

Common marmosets (Callithrix jacchus, n = 18) were trained to discriminate between rewarded and non-rewarded objects (simple discriminations, SDs) and to make conditional discriminations (CDs) when presented sequentially with two different pairs of identical objects signifying reward either in the right or left food well of the Wisconsin General Test Apparatus. After bilateral N-methyl-D-aspartate (0.12 M) lesions through the cornu ammonis-1 (CA1) field (7 microl in five sites), marmosets showed profound impairment in recall of CDs but not SDs, and were assigned to lesion only, lesion plus CA1 grafts and lesion plus Maudsley hippocampal cell line, clone 36 (MHP36) grafts groups matched for lesion-induced impairment. Cell suspension grafts (4 microl, 15-25 000 cells/microl) of cells dissected from the CA1 region of foetal brain at embryonic day 94-96, or of conditionally immortalized MHP36 cells, derived from the H-2Kb-tsA58 transgenic mouse neuroepithelium and labelled with [3H]thymidine, were infused at the lesion sites. The lesion plus MHP36 grafts group was injected five times per week with cyclosporin A (10 mg/kg) throughout testing. Lesion, grafted and intact control marmosets (n = 4-5/group) were tested on recall of SDs and CDs learned before lesioning and on acquisition of four new CDs over a 6-month period. Lesioned animals were highly impaired in recall and acquisition of CD tasks, but recall of SDs was not significantly disrupted. Both grafted groups of marmosets showed improvement to control level in recall of CDs. They were significantly slower in learning the first new CD task, but mastered the remaining tasks as efficiently as controls and were substantially superior to the lesion-only group. Visualized by Nissl staining, foetal grafts formed clumps of pyramidal-like cells within the denervated CA1 field, or jutted into the lateral ventricles. MHP36 cells, identified by beta-galactosidase staining and autoradiography, showed neuronal and astrocytic morphology, and were distributed evenly throughout the CA1 region. The results indicate that MHP36 cell grafts are as functionally effective as foetal grafts and appear to integrate into the host brain in a structurally appropriate manner, showing the capacity to differentiate into both mature neurons and glia, and to develop morphologies appropriate to the site of migration. These findings, which parallel the facilitative effects of foetal and MHP36 grafts in rats with ischaemic CA1 damage, offer encouragement for the development of conditionally immortal neuroepithelial stem cell lines for grafting in conditions of severe amnesia and hippocampal damage following recovery from cardiac arrest or other global ischaemic episodes.

Strychnine-sensitive glycine responses in neurons of the lateral amygdala: an electrophysiological and immunocytochemical characterization

Electrophysiological and staining techniques in the in vitro slice preparation of the rat and guinea-pig lateral amygdala were combined with immunocytochemical approaches, in order to characterize the neuronal substrate, the ionic basis and the pharmacological properties of glycine-mediated responses, and to map the distribution and composition of the mediating glycine receptors. Glycine was locally applied to spiny, pyramidal-like cells in the lateral amygdala, which possessed electrophysiological properties typical of projection neurons. Glycine induced a membrane hyperpolarization from rest and associated decrease in input resistance, and an interruption of spike firing and calcium-mediated high-threshold oscillations. The glycine-mediated response persisted during blocked synaptic transmission, reversed close to the presumed somatic chloride equilibrium potential and shifted during altered transmembrane Cl − gradients as expected for an increase in membrane chloride conductance. Responses to glycine were reversibly blocked by strychnine, but were insensitive to picrotoxin and bicuculline. Strychnine-sensitive components of spontaneous activity, but not of evoked synaptic responses, were frequently observed. Similar responses to glycine occurred in neurons of the guinea-pig and rat lateral amygdala, as well as in the central amygdala. The localization and composition of glycine receptors were examined through the use of monoclonal antisera directed against the binding protein (gephyrin), the α1 subunits (mAb2b) and α/ β subunits (mAb4a) of glycine receptors. A dense to moderate immunostaining for gephyrin was observed throughout the amygdaloid complex, whereas mAb4a immunofluorescent neurons, displaying strong punctate labelling around the soma and proximal dendrites, were confined to the lateral amygdala. No immunoreactivity was obtained with mAb2b antibodies in the amygdala. It is concluded that pyramidal-like projection cells in the lateral amygdala express functional glycine receptors at somatic and proximal dendritic sites, which are composed of β and α subunits other than the α1 type, and which may play a functional role in the control of excitatory activity in the amygdala, particularly during periods of decreased GABAergic influence.

Morphological and electrical characteristics of postnatal hippocampal neurons in culture: the presence of bicuculline- and strychnine-sensitive IPSPs

A modified method was developed for tissue-culturing postnatal hippocampal neurons using simple mechanical trituration for cell isolation and not including any hydrolysing enzymes, nerve growth factors or antiproliferating agents. The morphological properties of such neurons were characterized with light and interference polarizing microscopy, which revealed the appearance of growth cones from peripheral neurons and the presence of different types of neurons, including bipolar, stellate and pyramidal-like cells (i.e., pyramidal and dentate gyrus granule cells), which could be related to their putative counterparts in intact brain. The whole-cell configuration of the patch-clamp method was used for electrophysiological recordings of inhibitory synapses between these dissociated cultured neurons from the early postnatal rat hippocampus. This study indicated the presence of tetrodotoxin (TTX)-sensitive and TTX-resistant inhibitory postsynaptic potential (IPSPs) and inhibitory postsynaptic currents (IPSCs) in current-clamp and voltage clamp modes respectively. The coincident reversal potentials for IPSCs and for GABAA and glycine-evoked currents, and the sensitivity of the IPSCs to bicuculline or strychnine, indicated that these IPSCs were Cl−-dependent and mediated by either GABAA or glycine receptors. Inhibitory postsynaptic currents recorded under voltage-clamp conditions decayed with a time course that could be fitted by a single exponential with a value of 26 ms. An average quantal content of 2.5 was responsible for a typical GABA and glycine-activated IPSC and a single quantum for GABAergic input was inferred to activate about 160, and for glycinergic, about 200 Cl−, channels.

Mechanisms and functional significance of a slow inhibitory potential in neurons of the lateral amygdala

A slow inhibitory potential (sIP) elicited upon synaptic activation in spiny, pyramidal-like cells with properties indicative of projection neurons was investigated in slices of the rat and guinea-pig lateral amygdala in vitro. The sIP succeeded the triphasic sequence of excitatory and fast/slow inhibitory postsynaptic potentials mediated via glutamate and GABA(A/B) receptors, respectively, was readily evoked upon repetitive stimulation of the external capsule and appeared to terminate epileptiform burst discharges during pharmacologically reduced GABAergic influence. The sIP reversed close to the Cl- equilibrium potential, but was not affected by altered transmembrane Cl- gradients and not abolished by antagonists to ligand-gated Cl- channels. Intracellular injection of QX 314 and resulting blockade of sodium spikes had no effect, whereas the Ca2+ chelator BAPTA blocked the sIP concomitantly with slow hyperpolarizing afterpotentials following intrinsically generated spike firing, thereby indicating the contribution of Ca2+-dependent mechanisms secondary to synaptic activation. During action of BAPTA and QX 314, an N-methyl-D-aspartate (NMDA) receptor-mediated potential was unmasked, which contributed to the sIP. The Ca2+-dependent mechanisms of the sIP involved a membrane K+ conductance, as was indicated by the dependence on the K+ gradient and the shift of the reversal potential towards the K+ equilibrium potential during blocked NMDA receptors. During the presence of GABA receptor antagonists, reduction of the Ca2+-activated K+ conductance through injection of BAPTA or application of dopamine induced a gradual shift of interictal-like single bursts of spikes towards the generation of re-occurring ictal-like activity. It is concluded that pyramidal-like projection cells in the AL can generate a sIP upon synaptic activation, which reflects the combined activation of an NMDA receptor-mediated cation current and a K+ current that is secondary to the rise in intracellular Ca2+ concentration resulting from the preceding depolarizing response. The sIP may play an important role in controlling excitatory activity in the amygdala, particularly in preventing the transformation of interictal-like activity towards recurrent epileptic discharges during periods of decreased GABAergic influence.

Calretinin-immunoreactivity during postnatal development of the rat isocortex: a qualitative and quantitative study.

Postnatal development of the rat cortex is characterized by the gradual development of many calcium-dependent processes which demand a precise control of the intracellular levels of this cation; when the balance is disturbed, neuronal death ultimately ensues. Calretinin (CR), a calcium-binding protein, has been postulated to have neuroprotective capacity by buffering intracellular calcium. This putative relationship between CR and neuroprotection is still, however, a controversial issue. With a view to shedding further light on this subject, we studied the temporal and spatial distribution of CR in five different regions (the frontal- sensorimotor-, parietal-, temporal- and occipital region) of the rat cortex during postnatal development. Qualitative and quantitative immunocytochemistry of newborn, 5-, 10-, 15-, 20- and 30-day-old and adult rats revealed a profound increase in the density of the CR-positive neurons during the first two postnatal weeks in all regions examined. At the end of this period, CR-immunoreactive cells decreased sharply to adult levels. Cell classes exhibiting transient CR-immunoreactivity during the first two postnatal weeks included cells in layer I (amongst which were Cajal-Retzius cells), the subplate and pyramidal-like cells in the upper portion of layer V, most of them in the motor cortices. The above-described dynamics of CR expression were reflected also in the biochemical analysis performed (immunoblotting, ELISA). The temporal and spatial correlation with calcium-dependent events such as synaptogenesis, neurite elongation and remodelling in further support the contention that CR may play a neuroprotective role during postnatal development of the rat cortex.

Morphological characteristics of layer II projection neurons in the rat medial entorhinal cortex

The entorhinal cortex receives inputs from a variety of neocortical regions. Neurons in layer II of the entorhinal cortex originate one component of the perforant path which conveys this information to the dentate gyrus and hippocampus. The current study extends our previous work on the electroresponsive properties of layer II neurons of the medial entorhinal cortex in which we distinguished two categories of layer II neurons based on their electrophysiological attributes (Alonso and Klink [1993] J Neurophysiol 70:128–143). Here we report on the morphological features of layer II projection neurons, as revealed by in vitro intracellular injection of biocytin. We now report that the two electrophysiologically distinct types of neurons correspond to morphologically distinct types of cells. All neurons (65% of the total cells recorded) that developed sustained, subthreshold, sinusoidal membrane potential oscillations were found to have a stellate appearance. Neurons that did not exhibit oscillatory behavior had either a pyramidal-like (32%) or a horizontal cell morphology (3%). Stellate cells had multiple, thick, primary dendrites. Their widely diverging upper dendritic domain expanded mediolaterally over a distance of around 500 μm close to the pial surface. This mediolateral extent was more than double that of the pyramidal-like cells. Dendrites of stellate cells demonstrated long dendritic appendages, and their dendritic spines had a more complex morphology than those of nonstellates. The stellate cell axons emerged from a primary dendrite and were more than double the thickness (approximately 1.4 μm) of the axons of nonstellate cells. Recurrent axonal collaterization appeared more extensive in axons arising from stellate cells than from pyramidal-like cells. Hippocampus 1997;7:571–583. © 1997 Wiley-Liss, Inc.

Morphological characteristics of layer II projection neurons in the rat medial entorhinal cortex.

The entorhinal cortex receives inputs from a variety of neocortical regions. Neurons in layer II of the entorhinal cortex originate one component of the perforant path which conveys this information to the dentate gyrus and hippocampus. The current study extends our previous work on the electro-responsive properties of layer II neurons of the medial entorhinal cortex in which we distinguished two categories of layer II neurons based on their electrophysiological attributes (Alonso and Klink [1993] J Neurophysiol 70: 128-143). Here we report on the morphological features of layer II projection neurons, as revealed by in vitro intracellular injection of biocytin. We now report that the two electrophysiologically distinct types of neurons correspond to morphologically distinct types of cells. All neurons (65% of the total cells recorded) that developed sustained, subthreshold, sinusoidal membrane potential oscillations were found to have a stellate appearance. Neurons that did not exhibit oscillatory behavior had either a pyramidal-like (32%) or a horizontal cell morphology (3%). Stellate cells had multiple, thick, primary dendrites. Their widely diverging upper dendritic domain expanded mediolaterally over a distance of around 500 microns close to the pial surface. This mediolateral extent was more than double that of the pyramidal-like cells. Dendrites of stellate cells demonstrated long dendritic appendages, and their dendritic spines had a more complex morphology than those of nonstellates. The stellate cell axons emerged from a primary dendrite and were more than double the thickness (approximately 1.4 microns) of the axons of nonstellate cells. Recurrent axonal collaterization appeared more extensive in axons arising from stellate cells than from pyramidal-like cells.