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Background High extracellular glutamate concentrations have been identified as a likely trigger of epileptic seizures in mesial temporal lobe epilepsy (MTLE), but the underlying mechanism remains unclear. We investigated whether a deficiency in glutamine synthetase, a key enzyme in catabolism of extracellular glutamate in the brain, could explain the perturbed glutamate homoeostasis in MTLE.Methods The anteromedial temporal lobe is the focus of the seizures in MTLE, and surgical resection of this structure, including the hippocampus, leads to resolution of seizures in many cases. By means of immunohistochemistry, western blotting, and functional enzyme assays, we assessed the distribution, quantity, and activity of glutamine synthetase in the MTLE hippocampus.Findings In western blots, the expression of glutamine synthetase in the hippocampus was 40% lower in MTLE than in non-MTLE samples (median 44 [IQR 30–58] vs 69 [56–87]% of maximum concentration in standard curve; p=0·043; n=8 and n=6, respectively). The enzyme activity was lower by 38% in MTLE vs non-MTLE (mean 0·0060 [SD 0·0031] vs 0·0097 [0·0042] U/mg protein; p=0·045; n=6 and n=9, respectively). Loss of glutamine synthetase was particularly pronounced in areas of the MTLE hippocampus with astroglial proliferation, even though astrocytes normally have high content of the enzyme. Quantitative immunoblotting showed no significant change in the amount of EAAT2, the predominant glial glutamate transporter in the hippocampus.Interpretation A deficiency in glutamine synthetase in astrocytes is a possible molecular basis for extracellular glutamate accumulation and seizure generation in MTLE. Further studies are needed to define the cause, but the loss of glutamine synthetase may provide a new focus for therapeutic interventions in MTLE. [Copyright &y& Elsevier]

Background: High extracellular glutamate concentrations have been identified as a likely trigger of epileptic seizures in mesial temporal lobe epilepsy (MTLE), but the underlying mechanism remains unclear. We investigated whether a deficiency in glutamine synthetase, a key enzyme in catabolism of extracellular glutamate in the brain, could explain the perturbed glutamate homoeostasis in MTLE.Methods: The anteromedial temporal lobe is the focus of the seizures in MTLE, and surgical resection of this structure, including the hippocampus, leads to resolution of seizures in many cases. By means of immunohistochemistry, western blotting, and functional enzyme assays, we assessed the distribution, quantity, and activity of glutamine synthetase in the MTLE hippocampus.Findings: In western blots, the expression of glutamine synthetase in the hippocampus was 40% lower in MTLE than in non-MTLE samples (median 44 [IQR 30-58] vs 69 [56-87]% of maximum concentration in standard curve; p=0.043; n=8 and n=6, respectively). The enzyme activity was lower by 38% in MTLE vs non-MTLE (mean 0.0060 [SD 0.0031] vs 0.0097 [0.0042] U/mg protein; p=0.045; n=6 and n=9, respectively). Loss of glutamine synthetase was particularly pronounced in areas of the MTLE hippocampus with astroglial proliferation, even though astrocytes normally have high content of the enzyme. Quantitative immunoblotting showed no significant change in the amount of EAAT2, the predominant glial glutamate transporter in the hippocampus.Interpretation: A deficiency in glutamine synthetase in astrocytes is a possible molecular basis for extracellular glutamate accumulation and seizure generation in MTLE. Further studies are needed to define the cause, but the loss of glutamine synthetase may provide a new focus for therapeutic interventions in MTLE. [ABSTRACT FROM AUTHOR]

Background: The essential branched-chain amino acids (BCAAs) leucine, isoleucine, and valine have recently emerged as a potential novel treatment for medically refractory epilepsy. Blood-derived BCAAs can readily enter the brain, where they contribute to glutamate biosynthesis and may either suppress or trigger acute seizures. However, the effects of BCAAs on chronic (ie, spontaneous recurrent) seizures and epilepsy-associated neuron loss are incompletely understood., Materials and Methods: Sixteen rats with mesial temporal lobe epilepsy were randomized into 2 groups that could drink, ad libitum, either a 4% solution of BCAAs in water (n=8) or pure water (n=8). The frequency and relative percent of convulsive and nonconvulsive spontaneous seizures were monitored for a period of 21 days, and the brains were then harvested for immunohistochemical analysis., Results: Although the frequency of convulsive and nonconvulsive spontaneous recurrent seizures over a 3-week drinking/monitoring period were not different between the groups, there were differences in the relative percent of convulsive seizures in the first and third week of treatment. Moreover, the BCAA-treated rats had over 25% fewer neurons in the dentate hilus of the hippocampus compared with water-treated controls., Conclusions: Acute BCAA supplementation reduces seizure propagation, whereas chronic oral supplementation with BCAAs worsens seizure propagation and causes neuron loss in rodents with mesial temporal lobe epilepsy. These findings raise the question of whether such supplementation has a similar effect in humans.

Objective: In vivo studies of epilepsy typically use prolonged status epilepticus to generate recurrent seizures. However, reports on variable status duration have found discrete differences in injury after 40-50 min of seizures, suggesting a pathophysiologic sensitivity to seizure duration. In this report we take a multivariate cluster analysis to study a short duration status epilepticus model using in vivo 7T magnetic resonance spectroscopy (MRS) and histologic evaluation., Methods: The Hellier Dudek model was applied with 45 min of status epilepticus after which the animals were imaged twice, at 3 days and 3 weeks post-status epilepticus. Single voxel point resolved spectroscopy (PRESS) MRS was used to acquire data from the dentate gyrus and CA3 region of the hippocampus, assessing metabolite ratios to total creatine (tCr). In a subset of animals after the second imaging study, brains were analyzed histologically by Nissl staining., Results: A hierarchical cluster analysis performed on the 3-day data from 21 kainate-treated animals (dentate gyrus voxel) segregated into two clusters, denoted by KM (more injured, n = 6) and KL (less injured, n = 15). Although there was no difference in kainate dosing or seizure count between them, the metabolic pattern of injury was different. The KM group displayed the largest significant changes in neuronal and glial parameters; the KL group displayed milder but significant changes. At 3 weeks, the KL group returned to normal compared to controls, whereas the KM group persisted with depressed N-acetyl aspartate (NAA)/tCr, glutamate/tCr, and increased inositol/tCr and glutamine/tCr. The classification was also consistent with subsequent histologic patterns at 3 weeks., Significance: Although a short status period might be expected to generate a continuous distribution of metabolic injury, these data show that the short Hellier Dudek model appears to generate two levels of injury. The changes seen in segregated groups persisted into 3 weeks, and can be interpreted according to neuronal and glial biomarkers consistent with histology results., (© 2016 The Authors. Epilepsia published by Wiley Periodicals, Inc. on behalf of International League Against Epilepsy.)

Explosive blast shock waves and blunt impact to the head are two types of loading shown to result in mild traumatic brain injury (mTBI). While mTBI from these two causes shares some common features behaviorally, there are distinct differences in the pathophysiology of the underlying injury mechanisms. Various elucidations have been offered in the literature to explain the organic damage associated with mTBI resulting from both types of loading. The current state of understanding in this field is somewhat limited by the degree of appreciation of the physics and biomechanics governing the effects of explosive blast shock waves and blunt impact on the head, which has resulted in the various approaches to the investigation of the operative brain injury "wounding mechanisms". In this chapter we provide a simplified description of terminology associated with forces on the head from explosive blast shock waves and blunt impact, to assist readers in the field in evaluating interpretations of brain injury "wounding" processes. Remarkably, mTBI from either loading is shown generally to result in only a small loss of neurons, with hippocampal neurons appearing to be particularly vulnerable to explosive blast shock waves. Explosive blast studies in large animal models show a unique pattern of periventricular injury, which is different from the classic diffuse axonal injury. Both astrocyte and microglial activation are also seen in explosive blast as well as impact trauma, but this may be a general secondary brain injury response, nonspecific to explosive blast or blunt trauma. Additionally, while moderate to severe impact closed head injuries sometimes result in petechial hemorrhages or hematomas, they do not appear to be associated with explosive blast mTBI even with repeated exposure to blasts., (© 2015 Elsevier B.V. All rights reserved.)

Microinfusion of alpha2 adrenoreceptor agonists and antagonists into amygdala has contrasting effects on evoked and spontaneous seizure susceptibility in amygdala-kindled kittens. Subjects were 14 preadolescent kittens between 3 and 4 months old at the beginning of kindling. The same protocol was followed except that half the kittens received microinfusions (1 mul) of the alpha2 agonist clonidine (CLON; 1.32 nmol), and half received the alpha2 antagonist idazoxan (IDA; 0.33 nmol). Infusions were made over 1 min through needles inserted into cannulae adjacent to stimulating electrodes in the kindled amygdala, and evoked seizures were tested 10-12 min later. The results were: (1) CLON elevated seizure thresholds obtained once at the beginning and end of kindling, but only when compared to sham control values (needle insertion only) in the same animals; IDA significantly reduced thresholds. (2) CLON retarded and IDA accelerated kindling rate, defined as the number of afterdischarges (ADs) required to achieve the first stage 6 seizure or generalized tonic-clonic convulsion (GTC). These effects were most pronounced on the emergence of seizure "generalization" stages (3-6) from "focal" seizure stages (1-2). (3) CLON prevented onset of spontaneous seizures, whereas IDA precipitated onset of spontaneous seizures in 100% of the animals before or during the 5-week post-kindling follow-up during which seizures were evoked once each work day. The study confirms previous findings in kindled rodents to show that CLON and IDA can have opposing effects on kindling development in kittens and is the first report to show contrasting effects on spontaneous epileptogenesis in kindled animals as well.

Temporal lobe epilepsy (TLE) with hippocampal sclerosis is associated with high extracellular glutamate levels, which could trigger seizures. Down-regulation of glial glutamate transporters GLAST (EAAT1) and GLT-1 (EAAT2) in sclerotic hippocampi may account for such increases. Their distribution was compared immunohistochemically in non-sclerotic and sclerotic hippocampi and localized only in astrocytes, with weaker immunoreactivity for both transporters in areas associated with pronounced neuronal loss, especially in CA1, but no decrease or even an increase in areas with less neuronal loss, like CA2 and the subiculum in the sclerotic group. Such compensatory changes in immunoreactivity may account for the lack of differences between the groups in immunoblot studies as blots show the average concentrations in the samples. These data suggest that differences in glial glutamate transporter distribution between the two groups of hippocampi may be an insufficient explanation for the high levels of extracellular glutamate in sclerotic seizure foci observed through in vivo dialysis studies.

Purpose: Pathophysiologic mechanisms underlying temporal lobe epilepsy (TLE) are still poorly understood. One major hypothesis links alterations in energy metabolism to glutamate excitotoxicity associated with seizures in TLE. The purpose of this study was to determine whether changes in the activities of enzymes critical in energy and neurotransmitter metabolism contributed to the alterations in metabolic status leading to the excitotoxic effects of glutamate., Methods: Activities of four key enzymes involved in energy metabolism and glutamate cycling in the brain [aspartate aminotransferase (AAT), citrate synthase (CS), glutamate dehydrogenase (GDH), and lactate dehydrogenase (LDH)] were measured in anterolateral temporal neocortical and hippocampal tissues obtained from three different groups of medically intractable epilepsy patients having either mesial, paradoxical, or mass lesion-associated temporal lobe epilepsy (MTLE, PTLE, MaTLE), respectively., Results: We found that GDH activity was significantly decreased in the temporal cortex mainly in the MTLE group. A similar trend was recognized in the hippocampus of the MTLE. In all three patient groups, GDH activity was considerably lower, and AAT and LDH activities were higher in cortex of MTLE as compared with the corresponding activities in hippocampus (p<0.05). In the MTLE cortex and hippocampus, GDH activities were negatively correlated with the duration since the first intractable seizure., Conclusions: Our results support the hypothesis suggesting major alteration in GDH activity mainly in the MTLE group. It is proposed that significant alterations in the enzyme activities may be contributing to decreased metabolism of glutamate, leading to its accumulation.

Gamma knife radiosurgery is an attractive noninvasive treatment of brain tumors and vascular malformations that minimizes collateral tissue damage. However, exposure of normal tissue to even low-dose radiation triggers a cascade of acute and chronic injury and potentially significant morbidity and mortality. Because many irradiated patients now survive for years, identifying methods to prevent radiotherapy-induced collateral tissue damage is a major focus of current research. Erythropoietin (EPO), a cytokine produced locally by many tissues in response to injury, antagonizes apoptosis, reduces inflammation, and promotes healing. Systemic administration of recombinant EPO, widely used for treatment of anemia, provides robust protection from numerous insults in a variety of tissues, including the brain. Although irradiation injury is likely sensitive to EPO, the hematopoietic activity of EPO is undesirable in this setting, increasing erythrocyte number and predisposing to thrombosis. To avoid these potential adverse effects, we developed carbamylated EPO (CEPO) which does not stimulate the bone marrow. In this study, we show that CEPO (50 microg kg(-1) intraperitoneally) improves functional outcome when administered to adult rats just before, and then once daily for 10 d after, a necrotizing dose of radiation (100 Gy) to the right striatum. Immediately following irradiation, use and reflex movements of the contralateral forelimb to vibrissae stimulation were abnormal but rapidly improved in animals receiving CEPO. Moreover, histological examination revealed that the extent of brain necrosis after 90 days was reduced by approximately 50%. These findings further extend the kinds of injury for which administration of a tissue-protective cytokine provides benefit.

Purpose: Two subtypes of temporal lobe epilepsy (TLE) can be defined through clinical observations and analysis of hippocampal tissue resected during surgical procedures for intractable TLE: (a) mesial temporal sclerosis (MTS), which is characterized by extensive changes to the hippocampus and good surgical outcome; and (b) paradoxical temporal lobe epilepsy (PTLE), which is characterized by minimal cell loss and comparatively poorer surgical outcome. Patients in both subtypes have seizures that appear to begin in the medial temporal lobe, but documented differences in substrate and outcome between these subtypes has defined a need to distinguish MTS and PTLE patients before surgery. This report describes a retrospective study to investigate the feasibility of doing so during intracranial monitoring., Methods: Background EEG epochs, 5 min in duration, were recorded from the anterior hippocampus in 14 (10 MTS and four PTLE) patients with consistent localization of seizure onset to medial temporal structures. The power spectral density (PSD) of the EEG epochs was calculated by a Fourier spectral estimator, and the total signal power and power of the delta, theta, alpha, beta, and gamma frequency bands were submitted to group-to-group comparison., Results: Spectral peaks were observed in the delta band in all PSD estimates and in the theta band in nine of 14 (seven MTS, two PTLE) estimates. The MTS and PTLE subtypes could be distinguished by the total signal power and delta band power. These power measurements were greater in the PTLE subtype., Conclusions: Both delta and theta spectral components are present in hippocampal background EEGs recorded from patients with TLE. The results indicate that group differences exist in spectral measures of background hippocampal signals recorded from MTS and PTLE subtypes. This suggests both that substrate differences in cellular composition and connectivity are reflected in hippocampal background EEGs and that spectral measurements of these signals may hold promise for tests to identify the group membership of individual patients.

Erythropoietin (EPO), recognized for its central role in erythropoiesis, also mediates neuroprotection when the recombinant form (r-Hu-EPO) is directly injected into ischemic rodent brain. We observed abundant expression of the EPO receptor at brain capillaries, which could provide a route for circulating EPO to enter the brain. In confirmation of this hypothesis, systemic administration of r-Hu-EPO before or up to 6 h after focal brain ischemia reduced injury by approximately 50-75%. R-Hu-EPO also ameliorates the extent of concussive brain injury, the immune damage in experimental autoimmune encephalomyelitis, and the toxicity of kainate. Given r-Hu-EPO's excellent safety profile, clinical trials evaluating systemically administered r-Hu-EPO as a general neuroprotective treatment are warranted.

Mobile genetic elements termed transposons have been increasingly implicated in human disease. The small transposon mariner is widespread within non-vertebrate genomes and causes mutation by replication, excision, and insertion of itself without an RNA intermediate. We find that human DNA contains about 60 copies of this gene. Mariner transcripts are abundant in RNA prepared from sclerotic epileptic hippocampi. In contrast, typically no mariner-specific RNA is detected in non-sclerotic hippocampi from other epileptic patients or from autopsies. A complete but non-functional copy was obtained using rapid amplification of cDNA ends (RACE). This human mariner transcript is approximately 45% homologous to a functional counterpart active in Drosophila, with a coding region of 1035 bases flanked by 32 base inverted terminal repeats. The differential expression of mariner transcripts within sclerotic hippocampi suggests the probable activity of an autonomous element which by mutating critical genes could establish an epileptogenic substrate in the hippocampus.

The AMPA-type glutamate receptor subunits GluR1 and GluR2/3 were localized by immunohistochemistry with subunit-specific antibodies in hippocampi removed surgically from patients with temporal lobe epilepsy for the control of seizures. The flip and flop splice variants of the subunits were localized by in situ hybridization histochemistry with specific oligoprobes. In patient hippocampi that were not the seizure focus, the GluR1 subunit proteins were diffusely expressed on the dendrites of neurons in all regions. In contrast, in these same hippocampi, the GluR2/3 subunit proteins were expressed strongly on the soma and proximal dendrites of principal neurons in all regions. The flip variant of these subunits was localized in the hilus and fields of Ammon's Horn (CA), while the flop variants were prominent on the dentate granule cells. In the epileptogenic hippocampus, while immunoreactivity was decreased in all fields that showed neuronal loss, there was an increased expression of GluR1 on the dendritic excrescences on the proximal dendrites of hilar neurons and CA3 pyramidal neurons, as well as expression of GluR2/3 in hilar neuron excrescences. Electron microscopic examination confirmed that the GluR1 immunoreactivity was only in dendritic processes, particularly dense at the postsynaptic membranes. Such expression of GluR1 may provide for an enhanced glutamatergic response by these neurons. GluR2/3 was also significantly increased on the dendrites of dentate granule cells in the epileptogenic hippocampus and may provide some protection against excitotoxic injury by reducing calcium flux into neurons.

Purpose: We studied Na+ channel expression and the ability to generate action potential (AP)-like responses in primary cultures of human astrocytes by whole cell patch-clamp recording techniques., Methods: Tissue samples from 22 patients with various classifications of temporal lobe epilepsy (TLE) were plated to form separate astrocyte cultures from three regions; the hippocampus, parahippocampus, and anterolateral temporal neocortex., Results: The resting membrane potential of seizure focus astrocytes (MTLE, mesial TLE) was significantly depolarized (approximately -55 mV) as compared with cortical astrocytes (-80 mV). Hippocampal astrocytes from other substrates for TLE (MaTLE, mass-associated TLE; PTLE, paradoxical TLE) in which the hippocampus is not the seizure focus displayed resting membrane potentials similar to those of neocortical astrocytes (approximately -75 mV). Astrocytes from the seizure focus (MTLE) displayed much larger tetrodotoxin (TTX)-sensitive Na+ currents with -66-fold higher Na+ channel density (113.5 +/- 17.41 pA/pf) than that of comparison neocortical astrocytes (1.7 +/- 3.7 pA/pf) or than that of the hippocampal and parahippocampal astrocytes of the MaTLE and PTLE groups. As a consequence of this higher channel density, seizure focus astrocytes were capable of generating AP-like responses. However, at the resting potential, most Na+ channels are inactive and no spontaneous firing was observed. In contrast, astrocytes in the comparison neocortex from all groups and the hippocampus and parahippocampus from the MaTLE and PTLE groups could not fire AP-like responses even after large current injections., Conclusions: The function of Na+ channels in these astrocytes is unclear. However, the marked differences in seizure focus astrocytes as compared with cortical and nonseizure focus hippocampal astrocytes suggest a more active role for astrocytes associated with hyperexcitable neurons at a seizures focus.

The distribution of dynorphin (DYN), one of its binding sites (kappa 1 receptor) and their relationship to neuronal loss and granule cell hyperexcitability was examined in hippocampi from patients with temporal lobe epilepsy (TLE). In hippocampi that were not the seizure focus (mass associated temporal lobe epilepsy, MaTLE; and paradoxical temporal lobe epilepsy, PTLE) DYN-like immunoreactivity was localized in the dentate granule cells and their mossy fiber terminals within the hilus and area CA3. In hippocampi that were the seizure focus (MTLE), 89% showed an additional band of immunoreactivity confined to the inner molecular layer (IML) of the dentate gyrus, representing recurrent mossy fiber collaterals. In 11% of MTLE patients no staining was found in the IML (MTLE/DYN-). The MTLE/DYN- hippocampi were also characterized by a significantly lower degree of cell loss than in MTLE hippocampi in the dentate granule cell layer, the hilus and CA3. Both MTLE and MTLE/DYN- hippocampi showed evoked epileptiform bursting in granule cells while MTLE showed greater polysynaptic EPSPs and spontaneous excitatory activity. Thus granule cell recurrent collateral sprouting may account for only some aspects of hyperexcitability. In 30% of the MTLE group, hilar neurons of a variety of morphological types expressed DYN immunoreactivity in their somata and dendrites. The density of [3H]U69,593 binding sites in MaTLE and PTLE patients was highest in areas CA1 and the subiculum-regions having little or no DYN-staining. In the dentate molecular layer, hilus and CA3--regions with the most DYN immunoreactivity--there was a low density of ligand binding. The significance of this transmitter/receptor mismatch is yet unknown.

Medically intractable temporal lobe epilepsy is a common disease typically associated with hippocampal damage (sclerosis) and synaptic remodelling. These changes could include increased glutamate receptor expression, enhancing excitability and the potential for neuronal injury. We directly assessed this hypothesis using quantitative in vitro receptor autoradiography to determine the densities of glutamate-, NMDA-, quisqualate/alpha-amino-3-hydroxy-5-methyl-isoxazoleproprionic acid (AMPA)- and kainic acid-preferring binding sites in surgically removed hippocampi from patients with mesial temporal lobe epilepsy (sclerosis; MTLE) and patients with mass-associated temporal lobe epilepsy (no sclerosis; MaTLE), compared with autopsy material. Neuronal cell counts and in situ total protein densities were also obtained. In general, MaTLE and autopsy binding densities were indistinguishable. In contrast, some regions of MTLE hippocampi exhibited decreased receptor densities, with a corresponding loss of protein. In the hilus and CA1, however, ligand binding densities did not differ from the comparison groups in spite of markedly reduced protein content, consistent with increased glutamate receptor density. Kainate-preferring sites were distributed differently from the other glutamate subtypes and were uniformly decreased throughout the MTLE hippocampus, except for a unique expression within the outer dentate molecular layer. Along with increased NMDA and AMPA receptor densities in the hilus and CA1, this distinctive population of kainate receptors establishes that increased glutamate receptor expression is a feature of the remodelled MTLE hippocampus. These observations suggest that enhanced sensitivity to glutamate may be an important element in the pathophysiology of temporal lobe epilepsy.

Recent studies have established that the expression of defensive rage behavior in the cat is mediated over a descending pathway from the medial hypothalamus to the dorsolateral quadrant of the midbrain periaqueductal gray matter (PAG). The present study was designed to determine the roles played by 5-HT1A and 5-HT2/1C receptors in this region of PAG in modulating defensive rage behavior elicited from the cat's medial hypothalamus. Monopolar stimulating electrodes were implanted into the medial hypothalamus from which defensive rage behavior could be elicited by electrical stimulation. During the course of the study, the 'hissing' component of the defensive rage response was used as a measure of defensive rage behavior. Cannula-electrodes were implanted into sites within the PAG from which defensive rage could also be elicited by electrical stimulation in order that 5-HT compounds could be microinjected into behaviorally identifiable regions of the PAG at a later time. Microinjections of the selective 5-HT1A agonist, (+)-8-hydroxy-dipropylaminotetralin hydrobromide (8-OHDPAT) (50 pmol, 2.0 and 3.0 nmol), into the PAG suppressed the hissing response in a dose-dependent manner. Administration of the selective 5-HT1A antagonist, 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl] ethyl]-N-2-pyridinyl-benzamide hydrochloride (p-MPPI) (1.5 and 3.0 nmol), blocked the suppressive effects of 8-OHDPAT upon hissing. In contrast, microinjections of the 5-HT2/1C receptor agonist (+)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride ((+)-DOI hydrochloride) (0.01, 1.0 and 1.5 nmol) facilitated the occurrence of hissing elicited from the medial hypothalamus in a dose-dependent manner. Immunohistochemical analysis revealed the presence of 5-HT axons and preterminals throughout the PAG, and in particular, in its dorsolateral aspect which receives major inputs from the medial hypothalamus in association with defensive rage behavior. The overall findings of the study provide evidence that activation of 5-HT1A and 5-HT2/1C receptors within the midbrain PAG differentially modulate the expression of defensive rage behavior elicited from the medial hypothalamus of the cat.

Hippocampal dentate granule cells in temporal lobe epilepsy (TLE) patients with mesial sclerosis (MTLE) are reported to be hyperexcitable compared to those in patients with a mass lesion outside the hippocampus (MaTLE) (Williamson, Clin Neurosci 1994;2: 47-52). To determine if such hyperexcitability is associated with an altered morphology of these neurons, Lucifer Yellow-filled granule cells from MTLE patients were compared with those from MaTLE. The morphology of granule cells in both subject groups resembles closely that of human granule cells described previously by Golgi studies. About 40% of human granule cells have basal dendrites. Additionally their apical dendrites are much more limited in their spread in the longitudinal axis of the hippocampus contributing perhaps to a much more narrow lamellar organization than in rats. Analysis of variance computed on 21 morphometric parameters reveals a significant increase in the length of the portion of the dendrite in the inner molecular layer (IML), and a decrease in length in the outer third of the molecular layer in MTLE, compared to MaTLE. Factor analysis performed on the morphometric features of each group of neurons reveals that in the MaTLE neurons the most distinctive feature is the total dendritic length and the overall distribution of spines on them, whereas in MTLE a lengthening and elaboration of the dendrites in the IML is most distinctive. Previous observations of increased synaptic terminals containing neuropeptides, and neurotransmitter receptors in the IML taken in conjunction with an elaboration of granule cell dendrites in this region, suggest considerable synaptic reorganization within the IML of the MTLE hippocampus which may contribute to its epileptogenicity.

The distribution of the VIP receptor in the human hippocampus was studied by receptor autoradiography using [3-iodotyrosyl-125I]Vasoactive Intestinal Peptide (VIP) as a ligand, and the relationship of receptor distribution to the distribution of the peptide (visualized by immunocytochemistry) was examined in hippocampi surgically removed from patients with medically intractable temporal lobe epilepsy (TLE) and hippocampi obtained at autopsy from neurologically normal subjects. In the autopsy hippocampi and hippocampi from TLE patients with extrahippocampal temporal lobe lesions [125I]VIP binding was highest in the dentate molecular layer, with lower levels in the fields of Ammon's Horn (CA fields) and the subiculum. In hippocampi from patients with no temporal lobe lesions but considerable hippocampal neuronal loss there were significant elevations in the levels of ligand binding in all CA fields and the subiculum. Ligand binding densities in all CA fields of the patient hippocampi were strongly negatively correlated with neuronal numbers. Immunocytochemical localization of VIP shows no obvious change in the distribution patters of VIP immunoreactivity in the patient groups. This is the first demonstration of VIP and its receptor distribution in the human hippocampus. It is suggested that the elevated levels of receptor binding in the hippocampal seizure focus may indicate a mechanism for greater excitability of neurons and/or for their survivability in the face of the increased excitation and potential for injury in a seizure focus.

We demonstrate that the enzyme family responsible for the restoration of the transmembrane cation balance, namely the sodium pump (Na+, K(+)-ATPase), plays a critical role in whether glutamate injures adult neurons in vivo. Partial inhibition of the sodium pump by the cardiac glycoside ouabain in young adult rats is not itself damaging. This treatment, however, markedly potentiates ordinarily subtoxic dosages of the glutamate analog kainic acid to produce limbic seizures and widespread neurodegeneration within the hippocampus in a pattern closely resembling that observed for human temporal lobe epilepsy.

Na+,K(+)-ATPase (the sodium pump) is a ubiquitous enzyme that consumes ATP to maintain an adequate neuronal transmembrane electrical potential necessary for brain function and to dissipate ionic transients. Reductions in sodium pump function augment the sensitivity of neurons to glutamate, increasing excitability and neuronal damage in vitro. Temporal lobe epilepsy (TLE) is one disease characterized by hyperexcitability and marked hippocampal neuronal losses that could depend in part, on impaired sodium pump capacity secondary to changes in sodium pump levels and/or insufficient ATP supply. To assess whether abnormalities in the sodium pump occur in this disease, we used [3H]ouabain to determine the density of Na+,K(+)-ATPase for each anatomic region of hippocampus by in vitro autoradiography. Tissues were surgically obtained from epileptic patients with hippocampal sclerosis and compared with specimens from patients with seizures originating from temporal lobe tumors and autopsy controls. Changes in cellular population arising from neuronal losses or gliosis were assessed by protein densities derived from quantitative computerized densitometry of Coomassie-stained tissue sections. We estimated regional differences in capacity for ATP generation by determining cytochrome c oxidase (CO) activity. Principal neurons of hippocampus exhibit high levels of sodium pump enzyme. Both epilepsy groups exhibited slight but significant increases in sodium pump density/unit mass of protein in the dentate molecular layer, CA2, and subiculum as compared with autopsy controls. Greater hilar sodium pump density was also observed in sclerotic hippocampi. In contrast, CO activity was reduced in both epilepsy types throughout hippocampus. Results suggest that although sodium pump protein in surviving neurons appears to be upregulated in epilepsy, sodium pump capacity may be limited by the reduced levels of CO activity. Functional reduction in sodium pump capacity may be an important factor in hyperexcitability and neuronal death.

Patients with medically intractable temporal lobe epilepsy (TLE) undergo medial temporal lobectomy with hippocampectomy for one of two reasons. (1) A lesion (tumor or arteriovenous malformation) adjacent to, but not invasive of, the hippocampus, results in the removal of the lesion and adjacent hippocampus in order to ensure a tumor-free margin. This group will be referred to as tumor-related TLE (TTLE) patients. (2) The operation is performed when depth electrode recordings and other evaluative techniques point to the hippocampus as the focus of seizure initiation. This group will be referred to as cryptogenic TLE (CTLE) patients. Analysis of the hippocampi of these two groups of patients reveals that the TTLE hippocampus is quite similar to that of autopsy subjects in its chemical neuroanatomy. However, the dentate gyrus of the CTLE patients shows considerable morphological and cytochemical reorganization. This reorganization is characterized by a number of features. (1) There is a loss of granule cells which occurs either as a patchy loss and/or a thinning of the granule cell layer. (2) Remaining granule cells which contain dynorphin appear to produce recurrent collaterals into the inner molecular layer of the dentate gyrus. (3) In the subgranular region of the hilus (the polymorphic layer) there is a selective loss of interneurons immunoreactive for somatostatin, neuropeptide Y and substance P. (4) There appears to be an increase in fibers immunoreactive for somatostatin and neuropeptide Y which extend throughout the dentate molecular layer. Somatostatin fibers being less numerous than neuropeptide Y fibers (5). The distributions of a number of neurotransmitter receptors also show striking reorganization in the dentate gyrus of the CTLE hippocampus. (6) Second messenger systems protein kinase C and adenylate cyclase, and Na+, K(+)-ATPase activity, as determined by ouabain binding, is increased in the molecular layer of CTLE. This remodeling of the CTLE hippocampus may hold the key to the mechanisms of hyperexcitability of the granule cells in the hippocampus of this group, and consequently the generation of seizures. The removal of the hippocampus in CTLE patients results in good control of seizures, whereas removal of hippocampi that do not show such reorganization, in a group of patients classified as atypical CTLE patients, results in inadequate seizure control. These findings suggest a complex series of processes in converting the properly regulated granule cells into hyperexcitable ones.

Although neuropeptides have been demonstrated to be hippocampal neuromodulators in laboratory animals, their role in human hippocampal physiology or pathophysiology remains to be defined. The concentrations of somatostatin, cholecystokinin octapeptide, vasoactive intestinal polypeptide, and dynorphin A 1-17 were determined in hippocampal tissue resected from patients with cryptogenic temporal lobe epilepsy, a common seizure disorder originating in or near the hippocampus. Control tissue was obtained from cadavera or epilepsy patients in whom the hippocampus was removed during the resection of temporal lobe tumors. Peptide determinations were performed on extracts of punch biopsy specimens taken from six different hippocampal regions. A significant decrease in immunoreactive somatostatin concentration was identified in the dentate gyrus and in region cornu ammonis 4 of cryptogenic temporal lobe epilepsy specimens. No significant changes were present in any other hippocampal region or in the levels of other peptides. In situ hybridization studies performed on cryostat sections from similar patients confirmed a marked loss of neurons expressing the somatostatin gene, which was restricted to the dentate hilus. The density of specific 125I-somatostatin binding to cryostat sections, as determined by semiquantitative in vitro autoradiography, was significantly increased in the dentate gyrus of the cryptogenic epilepsy patients, compared with tumor control specimens. We conclude that a loss of somatostatin-producing interneurons with an upregulation of dentate somatostatin receptors is a specific and characteristic element in the pathophysiology of human cryptogenic temporal lobe epilepsy.

Electrical stimulation of the brain of the domestic cat elicited vocalizations from a site in the ventrolateral pons in the region of the medial lemniscus. The evoked vocalizations were analysed by means of sound spectrographs and classified as meows, growls, hisses and meow-growls. The neural pathways associated with these call sites were traced by following the pattern of fiber degeneration resulting from lesions placed at these sites. A descending fiber pathway was traced to the magnocellular tegmental field, the facial nucleus and the retrofacial nucleus, while an ascending system terminated in the zona incerta, the red nucleus, contralateral oculomotor nucleus, the ventroposterior lateral nucleus of the thalamus and inferior colliculus. It is concluded from these findings and the nature of the behavior evoked that the ventrolateral pontine call site lies on common pathway for a majority of vocalizations in the cat.

The ultrastructural organization of serotoninergic axons and terminals in the superficial dorsal horn of the monkey was examined by the PAP immunohistochemical method. Terminals with serotonin-like immunoreactivity (SLI) were identified in lamina I (marginal zone) and lamina IIo (outer substantia gelatinosa). Labelled profiles contained many small, round, clear vesicles and usually a few granular vesicles (70 nm diameter). Most synaptic junctions were symmetrical with sparse pre- and post-synaptic densities. Most frequently, terminals formed axodendritic synapses on large and small dendrites; axosomatic and axospinous contacts were infrequent. In addition SLI terminals were found apposed to unlabelled LGV-type terminals (containing several large granular vesicles of 75-90 nm). The appositions commonly met some criteria of axo-axonic synapses and the SLI terminal was suspected to be presynaptic. The unlabelled LGV terminal was often presynaptic to a dendrite, and it had characteristics similar to those observed for some primary afferents, particularly those which may contain substance P, a proposed transmitter for nociceptive C-fibers. Most of these 'triplet' complexes (SLI terminal apposing and LGV terminal synapsing onto dendrite) were found in the apical region of lamina I. The axodendritic and axosomatic serotoninergic contacts onto dorsal horn neurons may be a basis for some of the reported post-synaptic effects on dorsal horn cells of either local serotonin iontophoresis or of stimulation of the brainstem raphe, the probable origin of the serotoninergic terminals. These effects include both depression and excitation of the responses of the dorsal horn cells to electrical or natural stimulation of primary afferents, particularly C-fibers and nociceptors. Likewise, the contacts of SLI terminals with LGV terminals may provide a morphological substrate for the presynaptic effects also observed for serotonin iontophoresis or raphe stimulation, including changes in the excitability of primary afferent C-fibers.

Ultrastructural morphological relationship between microvessels and neural elements containing serotonin-like immunoreactivity, substance P and methionine-enkephalin like immunoreactivity were studied in the monkey and cat brain. A close relationship between serotonin-like immunoreactive dendritic processes, enkephalin-like immunoreactive profiles and capillary basement membrane was observed; they were usually separated by a double layer of glial membrane. More importantly, serotonin and substance P-like immunoreactive dendritic profiles are reported in close approximation with capillary basement membrane with no glial limitans at the site of contact. These intimate morphological associations may be the basis for neural regulation of tonicity, permeability and chemosensor function in the brain microvasculature.

Nest-coo (courtship) vocalization is an estrogen-dependent component of a sequence of behaviors leading to egg-laying in the ring dove (Streptopelia risoria). Implantation of estrogen in the nucleus intercollicularis of ovariectomized females restores the ability to produce nest-coos. Lesion of n. intercollicularis specifically disrupts nest-cooing and inhibits follicular development. We used anterograde transport of tritiated leucine to identify regions that receive input from n. intercollicularis. As a control, isotope was injected in an auditory nucleus (nucleus mesencephalicus lateralis, pars dorsalis) neighboring the n. intercollicularis. A strong projection to the nucleus was observed, demonstrating a potential interaction between auditory and vocalization systems at the level of the midbrain. Local projections of n. intercollicularis were observed in midbrain and hind brain nuclei including the nucleus tegmenti pedunculo-pontinus, pars compacta and nucleus papillioformis. A conspicuous projection ascended centrally via the mesencephalic central gray. Proceeding rostrally, this pathway was traced to the nucleus periventricularis magnocellularis and tissue surrounding it. At its rostral extent, the pathway was found to project to the nucleus preopticus medialis, the anterior medial hypothalamus, and the lateral hypothalamus. These results are consistent with the possibility of a nucleus intercollicularis projection to regions that regulate reproductive function.

Midbrain control of vocalization was evaluated in the ring dove by determining the major afferent inputs with retrograde tract tracing technique. Horseradish peroxidase was infused into various portions of the nucleus intercollicularis, an estrogen concentrating area, which disrupts nest calls when lesioned and induces the vocalization when stimulated by estrogen. Most labelled cell bodies were found in the archistriatum, including a region homologous to the mammalian amygdala.

The distribution of substance P-like (SPLI) and methionine-enkephalin-like (MELI) immunoreactivity in the thoracic, lumbar, and sacral human spinal cord was studied in sections stained by the indirect antibody peroxidase-antiperoxidase method. The laminar distributions of SPLI and MELI were somewhat similar. Immunoreactive axons and terminal-like processes were found in the marginal zone and substantia gelatinosa, although SPLI was heavier than MELI in Lissauer's tract and the marginal zone. Laterally, both SPLI and MELI extended along the entire dorsal horn border, including laminae IV and V. Isolated, fine fiber bundles also extended deep into the central regions of laminae IV and V. Heavy SPLI and MELI also were found in the intermediolateral column region (IML), while lesser amounts were observed in the intercalatus and intermediomedial (IMM) regions of lamina VII. In lamina IX, SPLI was moderate, and less MELI was found. The distribution of SPLI fibers and terminal-like profiles in the human spinal cord is similar to that in other mammals, whereas that of MELI suggests some differences, particularly in laminae I, II, and III. Many terminal-like structures were in close apposition to neurons in several laminae. Based upon correlations of the three-dimensional organization of both the labeled terminals and the dendritic trees of the cells, the types of neurons in apposition to SPLI and MELI could be tentatively identified. These may include (1) marginal cells resembling the pyramidal and multipolar types; (2) gelatinosal cells resembling islet and stalked cells; (3) spinothalamic cells in laminae I, V, VI, VII, and VIII; (4) autonomic neurons in the IML and IMM; and (5) motoneurons in lamina IX. In the sacral cord, neurons resembling dorsal band (lamina V) and lateral band (lamina VII) parasympathetic neurons also were outlined by SPLI and MELI, with more SPLI in the region of the lateral band neurons. Often, in many of the laminae, the same neuron was surrounded by both SPLI and MELI. The variety of possible neuronal types supplied by SPLI and MELI processes and the fairly wide distribution of these substances within the spinal cord suggest that they may be involved in a number of spinal functions.

Bilateral intercollicular lesions in the chick abolish or depress not only calling, but also those phases of behaviour when calling would have been occurring. These include: long bouts of excited feeding immediately after food is made available; examining and pecking moving targets and novel objects; persistent scanning, and inhibition of other behaviour in a novel environment. Deaf birds behave precisely like controls, so that possible auditory deficits are not involved. During calling phases significant visual stimuli are treated as if they were starting or conspicuous. Conversely, continuous examination of a stimulus causes calling to diminish or disappear even though response continues; a brief period when the stimulus is not seen causes calling to begin again when it is once more perceived. In addition to the increased effectiveness of relevant visual stimuli, motor facilitation is usual in calling phases, as is inhibition of irrelevant responses. Emotional behaviour in man and other mammals is compared to calling phases in the chick.

The indirect antibody-peroxidase immunostaining method was used to study the distribution of substance P- (SPLI), Met-enkephalin- (MELI) and serotonin-like (5-HTLI) immunoreactivity within the interpeduncular nuclear complex (IPN) of the cat. Populations of immunoreactive cell bodies were found to have a distinct pattern of localization within the IPN. This distribution pattern is described as seen at 4 representative levels rostrocaudally through the IPN. SPLI cell bodies were mainly at rostral levels I, II and III especially in the median region and as a cluster of cells in the intermediate regions of the IPN. MELI cell bodies were seen at all 4 levels, confined mainly to the median and intermediate regions at levels I and II and also in the lateral regions at levels III and IV. There were few 5-HTLI cell bodies at the rostral levels I and II where they were confined to the dorsal zone of the median region, whereas at levels III and IV there were numerous cell bodies located throughout the intermediate region of the IPN. The immunoreactive cell bodies in the IPN may belong to both intrinsic and projection neurons. The distinctive neurochemical cytoarchitectonics of the IPN suggest that this region of the brain which lies along the course of major limbic pathways, may be a center of complex integrative mechanisms of neuronal activity.

The localization of substance P-like immunoreactivity in the interpeduncular nucleus using the peroxidase-antiperoxidase technique, revealed that the median region of the interpeduncular nucleus was one area rich in substance P-like immunoreactive processes. The ultrastructural characteristics of these substance P-like immunoreactive processes and their organization within the middle zone of the median region of the interpeduncular nucleus was studied. Substance P-like immunoreactivity was found in the perikaryon of small neurons, and in proximal and small dendrites. The substance P-like immunoreactive cell bodies and proximal dendrites receive a variety of unlabeled synaptic terminals. The immunoreactive small dendrites usually formed the central component of a "rosette"-like formation with unlabeled terminals. A few immunoreactive small unmyelinated axons and boutons were also present in the neuropil. The substance P-like immunoreactive boutons contained mainly small round vesicles with some large dense-core vesicles. These substance P-like immunoreactive boutons were presynaptic to unlabeled dendritic profiles, and frequently to substance P-like immunoreactive dendritic profiles. They were also seen in apposition to unlabeled boutons. Substance P-like immunoreactive boutons were not found to synapse with the crest-like dendritic processes in this part of the interpeduncular nucleus. It is suggested on the basis of morphological features, that some of the unlabeled terminals synapsing on substance P-like immunoreactive dendrities, may be cholinergic in nature.

Bilateral lesions confined to the anterior intercollicular area (ICo) produce muting in domestic chicks. Calling is lost both in open field test, and to novel stimuli which evoke pecking. Lesions of the medial edge of the ICo also mute, perhaps by cutting connections with the central mesencephatic grey. Partial destruction of the ICo allows full intensity calling but with greatly increased latency. If an area ventral to the ICo is also destroyed, one type of calling reappears: it is still not readily evoked by particular stimuli which are effective in normal chicks but it often occurs during locomotion. Crowing survives ICo lesions and presumably depends on a different route to lower centres. Totally deaf birds show quite normal calling, so that hearing deficits are probably not involved in the muting. Finally, mammalian homologues of the ICo and its connections are identified.

In this study, we have identified over 150 cells in the adult macaque spinal cord that are immunoreactive when stained with an antibody to 5-HT by means of the PAP method. The staining was blocked by preabsorption of the primary antibody by the 5-HT-BSA conjugate (used to generate the antibody) and by 5-HT, while BSA, dopamine, norepinephrine, 5-methoxytryptamine and tryptamine preabsorption did not block staining. In addition, the Falck-Hillarp histofluorescence technique was applied to the lumbosacral cord from one of the three monkeys studied; results confirm the presence of cells with yellow fluorescence and rapid fading to brown characteristic for serotoninergic cells. The neuronal identity of these immunoreactive cells is based on light and electron microscopic morphology and the presence of synaptic terminals in contact with labeled somata and dendrites. Most of these neurons were small (10-25 micrometers) and located ventral to the central canal in lamina X. Some processes of the cells extended into the intermediate gray and the ventromedial area of the ventral horn; other processes wrapped around large blood vessels in lamina X or around the wall of the central canal. Cells were most frequent in the cervical cord (approximately 6-7/mm length of cord) and less frequent in the thoracic (1.5/mm), lumbar (3/mm), and sacral (2/mm) cord. A few cells were also found in the marginal and gelatinosa regions of the dorsal horn of the sacral cord. Examination of sections from the medulla-spinal cord junction (obex level) indicates that the spinal cells may be an extension of cell groups located near the raphe obscurus in the gray region around the IVth ventricle. The indolamine spinal cells may act as interneurons in spinal circuits, control spinal blood flow through vessel innervation, or play a role in CSF composition through central canal innervation.