Contralateral Hilus Research Articles

Post-Traumatic Hypoxia Exacerbates Neuronal Cell Death in the Hippocampus

Hypoxia frequently occurs in patients with traumatic brain injury (TBI) and is associated with increased morbidity and mortality. This study examined the effects of immediate or delayed post-traumatic hypoxia (fraction of inspired oxygen [FiO(2)] 11%) on acute neuronal degeneration and long-term neuronal survival in hippocampal fields after moderate fluid percussion injury in rats. In Experiment 1, hypoxia was induced for 15 or 30 min alone or immediately following TBI. In Experiments 2 and 3, 30 min of hypoxia was induced immediately after TBI or delayed until 60 min after TBI. In Experiment 1, acute neurodegeneration was evaluated in the hippocampal fields 24 h after insults using Fluoro-Jade staining and stereological quantification. During hypoxia alone, or in combination with TBI, mean arterial blood pressure was significantly reduced by approximately 30%, followed by a rapid return to normal values upon return to pre-injury FiO(2). Hypoxia alone failed to cause hippocampal neuronal degeneration when measured at 24 h after insult. TBI alone resulted in neuronal degeneration in each ipsilateral hippocampal field, predominantly in CA2-CA3 and the dentate gyrus. Compared to TBI alone, TBI plus immediate hypoxia for either 15 or 30 min significantly increased neuronal loss in most ipsilateral hippocampal fields and in the contralateral hilus and dentate gyrus. In Experiment 2, TBI plus hypoxia delayed 30 min significantly increased degeneration only in ipsilateral CA2-CA3. In Experiment 3, 30 min of immediate hypoxia significantly reduced the numbers of surviving neurons in the CA3 at 14 days after TBI. The greatly increased vulnerability in all hippocampal fields by immediate 30 min post-traumatic hypoxia provides a relevant model of TBI complicated with hypoxia/hypotension. These data underscore the significance of the secondary insult, the necessity to better characterize the range of injuries experienced by the TBI patient, and the importance of strictly avoiding hypoxia in the early management of TBI patients.

Changes in the patterns of care for small-cell-lung cancer (SCLC): Results of the 99–01 Patterns of Care Study (PCS) nationwide survey in Japan

6085 Background: Several key studies for SCLC preceded 2nd PCS nationwide survey in Japan. This study was undertaken to analyze the process of thoracic radiotherapy (TRT) and to evaluate changes in the patterns of care for SCLC. Methods: Between July 2002 and August 2004, the PCS conducted a second nationwide audit survey of care process for stage I-III SCLC patients treated with TRT between 1999–2001. PCS investigated; 1) patient background, 2) work-up studies, 3) process of RT, and 4) process of chemotherapy. Practice patterns of 99–01 PCS were compared with those of 95–97 PCS. Results: By using two-stage cluster sampling, the PCS expert team collected data for 139 eligible SCLC patients (men to women ratio, 5:1; median age, 69; age>70, 43%; KPS>70, 73%; stage III, 89%) from 73 institutions. Pre-treatment work-up study included chest CT in 96%, fiberoptic scope in 93%, brain CT or MRI in 86%, bone scinti in 79%. The median total dose of TRT was 5000 cGy. Twice-daily radiotherapy (BID) was used in 43%. The median field size of TRT was 12 cm x 14 cm. TRT port included ipsilateral hilus in 96%, ipsilateral mediastinum in 96%, contralateral mediastinum in 84%, and contralateral hilus in 17%. Field reduction during TRT course in 61%. The most predominantly used photon energy was 10 MV (77%), whereas obsolete technique using Co-60 or X-ray energy < 6MV comprised 12%. 3D-conformal therapy was used in 12%. Dose prescription was at an isodose line in 15%. CT-simulation was performed in 40%. Only 12 patients (9%) received prophylactic cranial irradiation (PCI). Ninety-two percent received systemic chemotherapy, of those, platinum based chemotherapy constituted 98%, and 73% were treated by concurrent chemoradiation (CCRT). Treatment by IRB-approved protocol was only 6 cases (4%). Compared with the previous 95–97 PCS, in the management of SCLC, significant increases in the use of CCRT (73% vs 37%, P<0.0001), BID-TRT (43% vs 19%, P<0.0001), and PCI (9% vs 2%, P=0.01) were observed, although the absolute number of patients receiving PCI was still extremely low. Conclusions: Evidence based CCRT and BID-TRT for SCLC had well penetrated into clinical practice, however, PCI has not yet widely accepted in Japan. No significant financial relationships to disclose.

Radiation therapy for small-cell lung cancer: results of the 1995–1997 patterns of care process survey in Japan

The Patterns of Care Study (PCS) conducted a nationwide audit survey in order to establish the national practice process of radiation therapy for small-cell lung cancer (SCLC) and examined the influence of institutional stratification on the process of care in Japan. The PCS randomly sampled institutions and patients using a two-stage cluster method and surveyed the process of radiation therapy for 174 stage I–III SCLC patients according to the category of institution, stratified as follows: A1, academic institutions treating ≥300 patients a year; A2, <300 patients; B1, non-academic institutions treating ≥120 patients a year; and B2, <120 patients. Karnofsky performance status distributions showed significant variance by stratification of institutions ( P=0.013). Patients treated on an outpatient basis accounted for 32% in A1, 23% in A2, 8% in B1, but only 5% in B2 ( P=0.007). A photon energy ≥10 MV was used for 87% of patients in A1, 69% in A2, 54% in B1 and 23% in B2 ( P=0.001). Contralateral hilus was irradiated for 11% of patients in A1, 17% in A2, 29% in B1 and 3% in B2 ( P=0.001). Field size reduction during the treatment course was done for 77% of patients in A1, 54% in A2, 60% in B1 and 42% in B2 ( P=0.007). Ninety-two percent of patients received combined chemotherapy and radiation therapy, and the most frequently used drugs were etoposide (91%) and cisplatin (69%). The results of clinical studies on SCLC had favorably penetrated into the clinical practice. However, the stratification of institutions significantly affected the process of radiation therapy in Japan.

Effect of seizures on hippocampal peptidergic neurons

Results from animal studies and from human tissue removed from epileptics show that certain subgroups of hippocampal neurons are more vulnerable to seizure activity than others. It is possible that neurons which contain calcium-binding proteins, such as parvalbumin, may be protected from the high calcium overload that results from seizure activity. In the present study, seizures were induced by an injection of tetanus toxin into the rat hippocampus. A morphological and quantitative analysis was made of the parvalbumin-containing neurons and of those which co-localized somatostatin and neuropeptide Y. At 2 weeks there was a generalized increase in immunoreactivity in both groups of neurons. From 1 month through to 3 months after injection, the up-regulation in immunoreactivity was sustained in the surviving hilar neurons which co-localized somatostatin and neuropeptide Y but there was a marked reduction in immunoreactivity of the parvalbumin neurons. Although there was no evidence for a loss of parvalbumin neurons there was a small and significant reduction in the number of somatostatin + neuropeptide Y double-labelled neurons in the contralateral hilus at 3 and 4 months after a tetanus injection. The vulnerability of the somatostatin + neuropeptide Y double-labelled hilar neurons but not of the parvalbumin-containing, presumed, basket cells are considered in terms of their connectivity.

Differential NPY mRNA expression in granule cells and interneurons of the rat dentate gyrus after kainic acid injection

Using in situ hybridization histochemistry neuropeptide Y (NPY) mRNA expression was investigated after intraperitoneal injection of kainic acid (KA) and after local application of KA or quinolinic acid into the dentate gyrus of the rat. Enhanced concentrations of NPY mRNA were observed in interneurons of the hilus, including presumptive fusiform neurons and pyramidal-shaped basket cells already 4 hours after initiation of limbic seizures by KA (10 mg/kg, i.p.). Increased NPY expression persisted in neurons resistant to seizure-induced cell death (6-48 h after i.p. KA). Exceptionally high hybridization signals were found in interneurons of the hilus and the CA1 and CA3 sectors 8 months after KA-induced limbic seizures. In the granule cell layer only a transient but pronounced increase in NPY mRNA was observed 12-24 h after injection. Only moderate changes were observed in this cell layer at later intervals. Anticonvulsant treatment with thiopental, after a brief period of generalized seizures, prevented the increase in NPY mRNA in granule cells but not in interneurons. No change in NPY message was found also in granule cells of rats which responded with mild "wet dog shake" behavior but not with motor seizures to KA injection. Local injections of low doses of KA (0.05-0.2 nmol) or quinolinic acid (6.5-100 nmol) into the dentate gyrus of the hippocampus under deep thiopental anesthesia, after 24 h, resulted in increased concentrations of NPY message in interneurons of the ipsilateral, but not of the contralateral hilus and not in granule cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of field potentials evoked in CA1 by angular bundle stimulation in the rat

Anatomical data and in vitro electrophysiological data suggest that entorhinal cortical efferents monosynaptically excite CA1 pyramidal cells. In vivo stimulation of the angular bundle (AB) evokes an extracellular waveform, suggestive of a population spike. Previously, this waveform has been claimed to be activation of the direct entorhinal cortex to CA1 pathway and a volume-conducted response from the dentate gyrus. This study sought to distinguish between these two possibilities using several techniques in urethane anesthetized rats. A comparison of waveforms evoked in the dentate gyrus (DG) and in CA1 by a single-shock test stimulus to the AB revealed that the early positive-to-negative phase of the waveform recorded in CA1 correlated in time and amplitude to the population spike recorded in the DG. When the AB test stimulus was preceded by a single shock to the AB or to the contralateral hilus, the amplitudes of the DG population spike and the early phase of the CA1 waveform decreased equally. Current source density analysis of the response to the AB test stimulus revealed that early current sinks localized only to the DG. Finally, focal application of the local anesthetic tetracaine into CA1 did not decrease the amplitude of the waveform evoked by the AB test stimulus. Tetracaine, however, greatly decreased the population spike evoked in CA1 by stimulating the contralateral CA3. Together these data indicate that the early positive-to-negative phase of the waveform in CA1 evoked by the AB test stimulus reflects events in the DG and not direct activation of CA1 by entorhinal cortical afferents.

Neurophysiological characterization of commissurally projecting dentate neurons in the rat

Commissural neurons in the dentate hilus and in the deep dentate granule cell layer were recorded intracellularly in vivo, in conjunction with combined injection of the antrograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) at sites of electrical stimulation. Two hilar neurons responded with short latency antidromic spikes to stimulation of the contralateral dentate infrapyramidal molecular layer, but did not show any synaptic potentials, suggesting that these neurons do not receive commissural hilar input, either directly or indirectly, from the stimulating sites. On the other hand, 3 dentate-hilar border neurons responded to the contralateral hilar stimulation with antidromic spikes, excitatory postsynaptic potentials (EPSPs), orthodromic spikes, and inhibitory PSPs (IPSPs), suggesting a rich synaptic interaction both commissurally and locally in this region. No direct commissural inhibition was observed in any of the cells. PHA-L injection at the stimulation site indicated that commissural hilar axon terminals project to a limited region of the contralateral molecular layer in a lamellar fashion, and have only a sparse distribution in the contralateral hilus. The results indicate that rapidly conducting commissural neurons in the dentate gyrus are themselves inhibited in an indirect manner by commissural fibers.

Cholinergic innervation of hippocampal GAD- and somatostatin-immunoreactive commissural neurons.

This study describes the cholinergic innervation of chemically defined nonpyramidal neurons in the hilar region of the rat hippocampus. Cholinergic terminals were identified by immunocytochemistry employing a monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme, and the avidin-biotin-peroxidase (ABC) technique. Nonpyramidal neurons in the hilar region were characterized by immunostaining with antibodies against glutamate decarboxylase (GAD), the gamma aminobutyric acid (GABA)-synthesizing enzyme, and somatostatin (SS). The immunoreactivity to these antibodies was detected by using biotinylated secondary antibodies and avidinated ferritin as an electron-dense marker. This electron microscopic double immunostaining procedure enabled us to demonstrate that immunoperoxidase-labeled ChAT-immunoreactive terminals established symmetric synaptic contacts on the ferritin-labeled GAD- and SS-immunoreactive hilar cells. In additional experiments at least some of the GAD- and SS-immunoreactive hilar neurons were further characterized as commissural neurons by retrograde filling with horseradish peroxidase (HRP) following an injection of the tracer into the contralateral hilus. From these triple labeling experiments, we concluded that at least some GABAergic and somatostatin-containing neurons in the hilar region, which are postsynaptic to cholinergic terminals, project to the contralateral hippocampus. Together with previous studies on the cholinergic innervation of the hippocampus and fascia dentata, our present results thus demonstrate that different types of hippocampal cells, including GABAergic and peptidergic commissural neurons in the hilar region, receive a cholinergic input.

Septohippocampal and commissural pathways antagonistically control inhibitory interneurons in the dentate gyrus

Available evidence suggests that a portion of the septohippocampal pathway may form inhibitory synapses on inhibitory interneurons in the dentate gyrus. In contrast, a portion of the commissural input from the contralateral hilus may form excitatory synapses on inhibitory interneurons. To ascertain whether these pathways synapse onto a common population of interneurons, a series of pulses were applied and their effects on perforant path evoked, granule cell population spikes were measured. The population spike was markedly reduced when the perforant path pulse was preceded by a pulse to the contralateral hilus. This inhibition was markedly reduced, however, when a medial septal pulse was applied either prior to, or within 3 ms after the commissural pulse. The disinhibition was critically dependent on the temporal relationship between the medial septal and commissural pulse, and not on the medial septal-perforant path relationship. This finding suggests that the septohippocampal pathway inhibits the interneurons through which the commissural pathway is able to inhibit granule cells.

Functional plasticity in two afferent systems of the granule cells in the rat dentate area: Frequency-related changes, long-term potentiation and heterosynaptic depression

Monosynaptic evoked field potentials (MEFP) were recorded in the dentate gyrus of male Wistar rats upon stimulation of either the perforant path or the commissural system. While the perforant path potential exhibited in acute experiments a clear reversal point of the field excitatory postsynaptic potential (EPSP) and population spike when protruding the registration electrode from the hippocampal fissura to the hilus of the dentate gyrus, the simultaneously registered commissural potential elicited by stimulation of the contralateral hilus showed no reversal of the negative monophasic wave but merely an amplitude maximum 40 μm above the reversal point of the perforant path potential. Frequency-related changes of the MEFPs during short tetanic stimulation with 15 Hz both in acute and chronic experiments, revealed differences in the properties of the input systems in that the commissural potential exhibited a clear frequency potentiation whereas the perforant path potential showed frequency depression. Pronounced long-term potentiation of the perforant path potential induced by 4 trains of tetanizing stimuli and lasting up to 72 h was accompanied by a long-term heterosynaptic depression of the commissural potential for up to 7 days after tetanization. Both the different frequency-related changes of the inputs and the extremely long duration of the heterosynaptic depression are discussed with respect to their proposed functions in the mechanisms of functional plasticity.

An analysis of the increase in granule cell excitability accompanying habituation in the dentate gyrus of the anesthetized rat

Repeated low-frequency stimulation of perforant path results in a decrement in the population EPSP and population spike recorded in the hilus of the dentate gyrus. The EPSP decrement is accompanied, however, by an increase in the population spike height/ population EPSP slope relation, suggesting that an increase in granule cell excitability also occurs. The present experiments explored the mechanisms of this apparent increase in excitability using standard field potential recording techniques to assess perforant path input/output curves in rats anesthetized with sodium pentobarbital. Low-frequency homosynaptic stimulationn (512 pulses, 1 Hz) of the perforant path resulted in a decreased spike threshold and overall shift to the left of the function relating population spike height to EPSP slope. These changes were consistently produced, even when granule cell discharge was inhibited by conditioning stimulation of the contralateral hilus. On the other hand, low-frequency heterosynaptic (lateral perforant path) or antidromic (mossy fiber) driving of the granule cells only slightly increased the medial path spike/EPSP relation, and did not alter the spike threshold. The excitability shift accompanying habituation was qualitatively different from that associated with long-term potentiation, but these shifts did not summate. The interpretation which best explains these various results is that granule cell excitability is increased during low-frequency perforant path stimulation by a process of disinhibition, caused by habituation of perforant path excitatory synaptic drive onto feed-forward inhibitory interneurons.

Ultrastructure of commissural neurons of the hilar region in the hippocampal dentate gyrus

Previous studies have described the polymorph neurons in the hilus of the dentate gyrus at the light microscopic level and have indicated that many of those neurons are the cells of origin for both ipsilateral associational and commissural projections to the dentate gyrus. Because previous studies have not described the ultrastructural characteristics of the hilar neurons, we identified these features of the commissural neurons in the hilus. The method of retrograde transport of horseradish peroxidase (HRP) was utilized with a silver staining technique for HRP intensification. Two populations of labeled commissural neurons were observed in electron microscopic preparations of the contralateral hilus. One type consisted of cells with somata that exhibited round or oval nuclei with no intranuclear inclusions and formed symmetric axosomatic synapses. The main dendrites of those neurons were thick and tapering. In contrast, the other type of labeled neuronal soma had infolded nuclei containing intranuclear rods or sheets, displayed both symmetric and asymmetric axosomatic synapses, and had dendrites that were less thick and generally aspinous. In those same preparations, labeled commissural axon terminals formed synapses with dendrites and dendritic spines in the hilus and molecular layer and iwth somata in the granule cell layer. From the results of this study it appears that there are two distinct populations of commissural hilar neurons: one type resembles the morphology of the spiny CA3 pyramidal neuron, a type of excitatory projection cell, and the other type is similar to the dentate gyrus basket cell, a local circuit neuron associated with GABAergic inhibition. This latter cell type provides further support for the notion that some commissural neurons are inhibitory.

Commissural inhibition and facilitation of granule cell discharge in fascia dentata.

Stimulation of the contralateral hippocampus in the hilar region had a marked effect on granule cell excitability in the fascia dentata. The primary effect was to block the population spike that otherwise occurred in response to perforant path stimulation. In contrast, the size the excitatory post-synaptic potential component of the perforant path-evoked field potential was only slightly reduced. The population spike diminution began at short latency (3.5 msec), beginning at about 1.0 msec after the onset of the slow component of the potential evoked by the contralateral stimulus. The completeness and duration of this population spike diminution depended on the contralateral stimulus intensity. The maximum duration was less than 40 msec in unanesthetized rats and more than 100 msec in rats under pentobarbital anaesthesia. Bicuculline did not diminish the field potential evoked by the contralateral stimulus but it did prevent the stimulus from blocking the perforant path population spike and, instead, permitted a weak facilitation of the population spike. The normal reduction of the population spike was not mediated by recurrent inhibition, secondary to granule cell activation, since it occurred whether or not the granule cells were inhibited at the time of contralateral stimulation. These results imply that the initial main effect on fascia dentata granule cells of activity in the contralateral hilus is a gamma-aminobutyric-acid-mediated inhibition. This effect most probably involves commissural excitation of local inhibitory interneurons. The direct excitatory action of commissural fibers on granule cells, by comparison, is very weak.

Inhibitory modulation of long-term potentiation: Evidence for a postsynaptic locus of control

Long-term potentiation (LTP) of the excitatory synapses of the perforant path onto the granule cells of the fascia dentata was prevented, or greatly reduced in amount, by stimulation of the contralateral hilus, a source of the commissural afferents and an indirect source of granule cell inhibition. The LTP was reduced only when the onset of contralateral stimulation fell within 50 ms before and 1 ms after the onset of the perforant path conditioning train. Over this time interval, the ability of the volley from the contralateral hilus to block granule cell discharge (population spike) caused by the perforant path conditioning train varied, but not with a time course corresponding to the reduction of LTP. Although trains of impulses applied at high frequency (3–8 pulses at 400 Hz) were often able to block the perforant path LTP completely, single shocks strong enough to block the perforant path population spike were not sufficient to reduce LTP. The results support Marr's proposal that local inhibitory networks may set the threshold for producing long-term synaptic modification. They do not support Hebb's nor Uttley's proposal since the long-term synaptic modification did not depend on either the occurence or the absence of a postsynaptic action potential.