Memantine protects cholinergic and glutamatergic septal neurons from Aß1-40-induced toxicity.

The medial septal region (medial septum and diagonal band of Broca, MS/DB) controls hippocampal excitability and synaptic plasticity. MS/DB cholinergic neurons degenerate early in Alzheimer's disease (AD). The presence of MS/DB glutamatergic neurons that project to the hippocampus and are vulnerable to Aß suggests that excitotoxicity plays a role in AD septal degeneration and hippocampal dysfunction. To demonstrate the presence of excitotoxicity in Aß-induced septal damage, we compared rats injected with Aß1-40 into the MS/DB with animals treated with memantine prior, during and after Aß1-40 injections. Controls were injected with phosphate buffered saline (PBS). MS/DB cholinergic, glutamatergic and GABAergic neurons were immunochemically identified. The number of MS/DB neurons was estimated using stereology. Our results show that memantine blocks Aß1-40-induced septal damage and suggest that excitotoxicity plays a role in basal forebrain neurodegeneration.

Endocytic trafficking signals in KCNMB2 regulate surface expression of a large conductance voltage and Ca(2+)-activated K+ channel.

Large conductance voltage and calcium-activated K(+) channels play critical roles in neuronal excitability and vascular tone. Previously, we showed that coexpression of the transmembrane beta2 subunit, KCNMB2, with the human pore-forming alpha subunit of the large conductance voltage and Ca(2+)-activated K(+) channel (hSlo) yields inactivating currents similar to those observed in hippocampal neurons [Hicks GA, Marrion NV (1998) Ca(2+)-dependent inactivation of large conductance Ca(2+)-activated K(+) (BK) channels in rat hippocampal neurones produced by pore block from an associated particle. J Physiol (Lond) 508 (Pt 3):721-734; Wallner M, Meera P, Toro L (1999b) Molecular basis of fast inactivation in voltage and Ca(2+)-activated K(+) channels: A transmembrane beta-subunit homolog. Proc Natl Acad Sci U S A 96:4137-4142]. Herein, we report that coexpression of beta2 subunit with hSlo can also modulate hSlo surface expression levels in HEK293T cells. We found that, when expressed alone, beta2 subunit appears to reach the plasma membrane but also displays a distinct intracellular punctuated pattern that resembles endosomal compartments. beta2 Subunit coexpression with hSlo causes two biological effects: i) a shift of hSlo's intracellular expression pattern from a relatively diffuse to a distinct punctated cytoplasmic distribution overlapping beta2 expression; and ii) a decrease of hSlo surface expression that surpassed an observed small decrease in total hSlo expression levels. beta2 Site-directed mutagenesis studies revealed two putative endocytic signals at the C-terminus of beta2 that can control expression levels of hSlo. In contrast, a beta2 N-terminal consensus endocytic signal had no effect on hSlo expression levels. Thus, beta2 subunit not only can influence hSlo currents but also has the ability to limit hSlo surface expression levels via an endocytic mechanism. This new mode of beta2 modulation of hSlo may depend on particular coregulatory mechanisms in different cell types.

Electrophysiological and morphological heterogeneity of slow firing neurons in medial septal/diagonal band complex as revealed by cluster analysis.

Slow firing septal neurons modulate hippocampal and neocortical functions. Electrophysiologically, it is unclear whether slow firing neurons belong to a homogeneous neuronal population. To address this issue, whole-cell patch recordings and neuronal reconstructions were performed on rat brain slices containing the medial septum/diagonal band complex (MS/DB). Slow firing neurons were identified by their low firing rate at threshold (<5 Hz) and lack of time-dependent inward rectification (Ih). Unsupervised cluster analysis was used to investigate whether slow firing neurons could be further classified into different subtypes. The parameters used for the cluster analysis included latency for first spike, slow after-hyperpolarizing potential, maximal frequency and action potential (AP) decay slope. Neurons were grouped into three major subtypes. The majority of neurons (55%) were grouped as cluster I. Cluster II (17% of neurons) exhibited longer latency for generation of the first action potential (246.5+/-20.1 ms). Cluster III (28% of neurons) exhibited higher maximal firing frequency (25.3+/-1.7 Hz) when compared with cluster I (12.3+/-0.9 Hz) and cluster II (11.8+/-1.1 Hz) neurons. Additionally, cluster III neurons exhibited faster action potentials at suprathreshold. Interestingly, cluster II neurons were frequently located in the medial septum whereas neurons in cluster I and III appeared scattered throughout all MS/DB regions. Sholl's analysis revealed a more complex dendritic arborization in cluster III neurons. Cluster I and II neurons exhibited characteristics of "true" slow firing neurons whereas cluster III neurons exhibited higher frequency firing patterns. Several neurons were labeled with a cholinergic marker, Cy3-conjugated 192 IgG (p75NTR), and cholinergic neurons were found to be distributed among the three clusters. Our findings indicate that slow firing medial septal neurons are heterogeneous and that soma location is an important determinant of their electrophysiological properties. Thus, slow firing neurons from different septal regions have distinct functional properties, most likely related to their diverse connectivity.

Septal GABAergic neurons are selectively vulnerable to pilocarpine-induced status epilepticus and chronic spontaneous seizures.

The septal region of the basal forebrain plays a critical role modulating hippocampal excitability and functional states. Septal circuits may also play a role in controlling abnormal hippocampal hyperexcitability in epilepsy. Both lateral and medial septal neurons are targets of hippocampal axons. Since the hippocampus is an important epileptogenic area in temporal lobe epilepsy, we hypothesize that excessive excitatory output will promote sustained neurodegeneration of septal region neurons. Pilocarpine-induced status epilepticus (SE) was chosen as a model to generate chronic epileptic animals. To determine whether septal neuronal populations are affected by hippocampal seizures, immunohistochemical assays were performed in brain sections obtained from age-matched control, latent period (7 days post-SE) and chronically epileptic (more than one month post-SE survival) rats. An anti-NeuN (neuronal nuclei) antibody was used to study total neuronal numbers. Anti-ChAT (choline acetyltransferase), anti-GAD (glutamic acid decarboxylase) isoenzymes (65 and 67), and anti-glutamate antibodies were used to reveal cholinergic, GABAergic and glutamatergic neurons, respectively. Our results revealed a significant atrophy of medial and lateral septal areas in all chronically epileptic rats. Overall neuronal density in the septum (medial and lateral septum), assessed by NeuN immunoreactivity, was significantly reduced by approximately 40% in chronically epileptic rats. The lessening of neuronal numbers in both regions was mainly due to the loss of GABAergic neurons (80-97% reduction in medial and lateral septum). In contrast, populations of cholinergic and glutamatergic neurons were spared. Overall, these data indicate that septal GABAergic neurons are selectively vulnerable to hippocampal hyperexcitability, and suggest that the processing of information in septohippocampal networks may be altered in chronic epilepsy.

Potassium (K+) channel expression in basal forebrain cholinergic neurons.

Basal forebrain cholinergic neurons (BFCN) are depleted early in the course of Alzheimer's disease (AD). BFCN voltage-gated K(+) channels regulate acetylcholine release and may play a role in BFCN neurodegeneration. Neuronal voltage-gated K(+) channels are heterotetrameric assemblies of K(v) and accessory subunits. Currently, there is no available information about the K(v) proteins expressed in BFCN. Immunohistochemical techniques were used to investigate the expression of specific K(v) subunits in rat brain BFCN. Our results showed that BFCN express both K(v)3.1 and K(v)2.1 subunits. However, the K(v)2.1 subunit showed a wider distribution in noncholinergic neurons than the K(v)3.1 subunit. K(v)3.1 and K(v)2.1 immunostaining was noticeable not only in neuronal cell bodies but also in the dendritic ramifications of these neurons. Insofar as the K(v)3.1 subunit has been classically associated with "fast-spiking neurons" and BFCN have low firing rates and long-duration action potentials, K(v)3.1 subunits may have functions other than facilitating high-frequency firing in BFCN.

Mechanisms of neural synchrony in the septohippocampal pathways underlying hippocampal theta generation.

Using urethane-anesthetized rats, 18 simultaneously recorded septohippocampal cell pairs (36 individual cells), each classified as theta-related according to the criteria of, were studied during four spontaneously occurring hippocampal field conditions: (1) large amplitude irregular activity (LIA) only; (2) the transition from LIA to theta; (3) theta only; and (4) the transition from theta to LIA. The main objective was to study the temporal relationships and degree of neural synchrony between the discharges of the cell pairs, using both time-averaged and time-dependent joint peristimulus time histogram correlation techniques, during the four conditions, to determine their contribution to the control of oscillation and synchrony (theta) in the hippocampus. The study demonstrated that the transition from the LIA state to the theta field state in the hippocampus required a temporal sequence of changes in theta-related cellular activity occurring on average 500 msec preceding the transition: (1) the medial septum inhibits hippocampal theta-OFF cells; (2) medial septal tonic theta-ON cells provide tonic depolarizing inputs to initiate membrane potential oscillations (MPOs) in hippocampal phasic theta-ON cells, whereas medial septal phasic theta-ON cells synchronize the MPOs of hippocampal phasic theta-ON cells and the discharges of hippocampal tonic theta-ON cells. Much of the time preceding the LIA to theta transition is accounted for by recruitment of these theta-related cell populations. Conversely, "turning off" the theta state occurs abruptly and involves the medial septal disinhibition of hippocampal theta-OFF cells.

Role of potassium channels in amyloid-induced cell death.

Basal forebrain cholinergic neurons are severely depleted early in Alzheimer's disease and appear particularly susceptible to amyloid beta-peptide (A beta) toxicity in vivo. To model this effect in vitro, a cholinergic septal cell line (SN56) was exposed to A beta. SN56 cells exhibited a tetraethylammonium (TEA)-sensitive outward K+ current with delayed rectifier characteristics. Increases of 64% (+/-19; p < 0.02) and 44% (+/-12; p < 0.02) in K+ current density were noted 6-12 and 12-18 h following the addition of A beta to SN56 cell cultures, respectively. Morphological observation and staining for cell viability showed that 25 +/- 4 and 39 +/- 4% of SN56 cells were dead after 48- and 96-h exposures to A beta, respectively. Perfusion of SN56 cells with 10-20 mM TEA blocked 71 +/- 6 to 92 +/- 2% of the outward currents, widened action potentials, elevated [Ca2+]i, and inhibited 89 +/- 14 and 68 +/- 14% of the A beta toxicity. High [K+]o, which depolarizes cell membranes and increases [Ca2+]i, also protected SN56 cells from A beta toxicity. This effect appeared specific since glucose deprivation of SN56 cells did not alter K+ current density and TEA did not protect these cells from hypoglycemic cell death. Furthermore, A beta was toxic to a dopaminergic cell line (MES23.5) that expressed a K+ current with delayed rectifier characteristics; K+ current density was not altered by A beta and MES23.5 cells were not protected by TEA from A beta toxicity. In contrast, a noncholinergic septal cell line (SN48) that shows minimal outward K+ currents was resistant to the toxicity of A beta. These data suggest that a K+ channel with delayed rectifier characteristics may play an important role in A beta-mediated toxicity for septal cholinergic cells.

Modulation of septal cell activity by extracellular zinc.

Zinc released from axon terminals in the brain can interact with multiple membrane channels and receptors. However, the specific effects of these Zn(2+)-dependent interactions on physiological processes remains unclear. Because Zn(2+)-containing axon terminals are abundant in the septal region, we selected a septal cell line (SN56) to study the effects of Zn2+ on cell activity. Voltage-clamp recordings showed well-developed voltage-dependent Na+, Ca2+ and K+ currents. Micromolar concentrations of Zn2+ partially blocked Na+ and Ca2+ currents without affecting K+ currents. Current-clamp recordings showed that SN56 cells fire spontaneous and evoked action potentials. While most (> or = 83%) Na+ and Ca2+ currents were blocked with 1 microM tetrodotoxin (TTX) and 2 mM Co2+, action potentials persisted after either 1 microM TTX or 2 mM Co2+ application. In contrast, concentrations of Zn2+ (50-300 microM) that induced incomplete blockade (< or = 50%) of either Ca2+ and Na+ currents abolished action potential generation. These data show that simultaneous and partial blockade of Ca2+ and Na+ channels by Zn2+ inhibit SN56 cell activity. Because septal outputs extensively modulate the excitability of cortical and subcortical brain regions, Zn2+ inhibition of action potential generation in septal neurons could play an important physiological role in regulating brain activity.

Amyotrophic lateral sclerosis immunoglobulins increase intracellular calcium in a motoneuron cell line.

A hybrid motoneuron cell line (VSC4.1) was used as a model system to study the relationship between alterations in intracellular calcium and subsequent cell death induced by immunoglobulin fractions purified from sera of patients with ALS. Using fluo-3 fluorescence imaging, immunoglobulins from 8 of 10 patients with ALS were found to induce transient increases in intracellular calcium ([Ca2+]i) in differentiated VSC4.1 cells. These transient [Ca2+]i increases required extracellular calcium entry through voltage-gated calcium channels sensitive to synthetic FTX and to high concentrations (>1 microM) of omega-agatoxin IVa. The incidence of transient [Ca2+]i increases induced by ALS immunoglobulins correlated with the extent of cytotoxicity induced by the same ALS immunoglobulins in parallel cultures of VSC4.1 cells. Furthermore, manipulations which blocked transient [Ca2+]i increases (addition of synthetic FTX or omega-agatoxin IVa) also inhibited the cytotoxic effects of ALS immunoglobulins. No transient calcium increases were observed in VSC4.1 cells following addition of immunoglobulins from 7 neurologic disease control patients. However, transient [Ca2+]i increases were observed following addition of immunoglobulins from 4 of 5 patients with myasthenia gravis (MG). The [Ca2+]i changes induced by MG immunoglobulins were not blocked by s-FTX, suggesting that they result from a different mechanism than those induced by ALS immunoglobulins. These results suggest that immunoglobulins from patients with ALS can induce transient increases in intracellular calcium in a motoneuron cell line, which may represent early events in the cascade of processes leading to injury and death of susceptible cells.

Expression of calbindin-D28K in motoneuron hybrid cells after retroviral infection with calbindin-D28K cDNA prevents amyotrophic lateral sclerosis IgG-mediated cytotoxicity.

Calbindin-D28K and/or parvalbumin appear to influence the selective vulnerability of motoneurons in amyotrophic lateral sclerosis (ALS). Their immunoreactivity is undetectable in motoneurons readily damaged in human ALS, and in differentiated motoneuron hybrid cells [ventral spinal cord (VSC 4.1 cells)] that undergo calcium-dependent apoptotic cell death in the presence of ALS immunoglobulins. To provide additional evidence for the role of calcium-binding proteins in motoneuron vulnerability, VSC 4.1 cells were infected with a retrovirus carrying calbindin-D28K cDNA under the control of the promoter of the phosphoglycerate kinase gene. Differentiated calbindin-D28K cDNA-infected cells expressed high calbindin-D28K and demonstrated increased resistance to ALS IgG-mediated toxicity. Treatment with calbindin-D28K antisense oligodeoxynucleotides, which significantly decreased calbindin-D28K expression, rendered these cells vulnerable again to ALS IgG toxicity.

Apoptosis induced by beta-N-oxalylamino-L-alanine on a motoneuron hybrid cell line.

It has been suggested that beta-N-oxalylamino-L-alanine, a non-protein amino acid present in the Lathyrus Sativus seeds, may play a role in the etiopathogenesis of neurolathyrism, a toxic form of motor neuron disease clinically characterized by a severe spastic paraparesis. In order to investigate the mechanisms of beta-N-oxalylamino-L-alanine-mediated cell death, we studied the effect of this neurotoxin as well as other excitatory amino acids agonists on the growth and survival of motoneuron hybrid ventral spinal cord 4.1 cells. beta-N-oxalylamino-L-alanine was toxic to ventral spinal cord 4.1 cells in a concentration-dependent fashion (0.5-10 mM). Among the excitatory amino acids tested, only glutamate (1-10 mM), quisqualate (1 mM) and, with less extent, beta-N-methylamino-L-alanine (10 mM) induced a significant reduction of cell survival. The effect of Lathyrus Sativus neurotoxin was a slow process, becoming apparent only after 24-48 h of incubation. Interestingly, a mathematical analysis applied to the time course and dose curve of beta-N-oxalylamino-L-alanine toxicity suggested that even for very low concentrations of the amino acid it is theoretically possible to predict a time-dependent effect. The cell death was not blocked by antagonists of N-methyl-D-aspartate or non-N-methyl-D-aspartate receptors; aurintricarboxylic acid and alpha-tocopherol gave a partial protection; cysteine (1 mM) prevented the toxic effect of both Lathyrus Sativus neurotoxin and glutamate as well as quisqualate. Morphologically, in the presence of either beta-N-oxalylamino-L-alanine, glutamate or quisqualate, ventral spinal cord 4.1 cells showed apoptotic features also confirmed by ISEL technique and agarose gel electrophoresis of genomic DNA. Thus, our results suggest that in ventral spinal cord 4.1 motoneuron hybrid cells, in the absence of functional synaptic excitatory amino acid receptors, beta-N-oxalylamino-L-alanine induces cell degeneration through an apoptotic mechanism, possibly mediated by a block of cystine/glutamate Xc antiporter.

Ascending projections of the posterior nucleus of the hypothalamus: PHA-L analysis in the rat.

With the exception of a report by R.B. Veazey, D.G. Amaral, and W.M. Cowan (1982, J. Comp. Neurol. 207:135-156) that examined the projections of the posterior hypothalamic area in the monkey by using the autoradiographic technique, the ascending projections of the posterior nucleus (PH) of the hypothalamus have not been systematically examined in any species. The present report describes the ascending projections of PH in the rat by using the anterograde anatomical tracer, Phaseolus vulgaris-leucoagglutinin (PHA-L). The major ascending route for PH fibers is the medial forebrain bundle. PH fibers project densely to several subcortical and cortical sites. The subcortical sites are the subthalamus/hypothalamus (zona incerta, the supramammillary nucleus, lateral, perifornical, dorsal, and anterior nuclei/areas), the thalamus (lateroposterior, laterodorsal, parafascicular, reuniens, paraventricular, central medial, paracentral, central lateral and intermediodorsal nuclei), the amygdala (central, lateral, and medial nuclei), the septal area (bed nucleus of stria terminalis, medial and lateral septum), and the basal forebrain (horizontal/vertical limbs of diagonal band nuclei and lateral preoptic area). The cortical sites are the perirhinal, insular, frontal (lateral agranular), prelimbic, and infralimbic cortices. The diversity of PH projections to subcortical and cortical "limbic-related" sites and to several structures with direct input to the hippocampus (supramammillary nucleus, reuniens, paraventricular and laterodorsal nuclei of the thalamus, medial and lateral septum, and perirhinal cortex) suggest that the PH may serve a critical role in various components of emotional behavior, including mnemonic processes associated with significant emotional events.

Cell death induced by beta-amyloid 1-40 in MES 23.5 hybrid clone: the role of nitric oxide and NMDA-gated channel activation leading to apoptosis.

The molecular events associated with beta-amyloid-induced neuronal injury remain incompletely characterized. Using a substantia nigra/neuroblastoma hybrid cell line (MES 23.5) synthetic beta-amyloid 1-40 induced a time and dose-dependent apoptotic cell death which was characterized by cell shrinkage and fragmentation of DNA, and was inhibited by aurintricarboxylic acid (ATA), and cycloheximide (CHX). Following beta-amyloid 1-40 treatment, cyclic GMP, an index of NO synthesis, was increased in MES 23.5 cells. The NO scavenger hemoglobin, as well as the NO synthase inhibitors NG-monomethyl-L-arginine acetate (L-NMMA) and L-N5-(1-iminoethyl)ornithine hydrochloride (L-NI0) attenuated such increases. These same inhibitors and scavengers also significantly prevented cytotoxicity. beta-Amyloid also induced an early and transient increase in intracellular calcium as monitored with laser scanning confocal microscopy and Fluo-3 imaging. These induced calcium transients could be significantly blocked by the N-methyl-D-aspartic acid (NMDA) receptor antagonist MK-801. Pretreatment with MK-801 or removal of extracellular Ca2+ also reduced beta-amyloid-induced NO production and neurotoxicity. Furthermore, beta-amyloid neurotoxicity was greatly enhanced in the absence of Mg2+ or in the presence of glutamate or NMDA. These data suggest that beta-amyloid can lead to apoptotic cell death through a NO mediated process possibly triggered by Ca2+ entry through activated NMDA-gated channels.

Increased intracellular calcium triggered by immune mechanisms in amyotrophic lateral sclerosis.

Although the causes of motor neuron degeneration and death in amyotrophic lateral sclerosis (ALS) is unknown, recent evidence suggests a prominent role for increased intracellular calcium, possibly triggered by autoimmune mechanisms. The presence in ALS patients of paraproteinemias, lymphomas, lymphoid cells in the central nervous system (CNS) and the availability of animal models of immune-mediated motor neuron disease provide circumstantial evidence for autoimmunity. Direct evidence derives from the demonstration that ALS IgGs bind to voltage-gated calcium channels in 75% of sporadic cases, but not in familial ALS cases, and that ALS IgGs increase N-type and P-type calcium currents in neuronal cells and in lipid bilayers. These same ALS IgGs are cytotoxic for a motor neuron cell line (VSC 4.1) in vitro. In addition, following passive transfer to mice in vivo, ALS IgGs produce ultrastructural and calcium changes in synaptic vesicles and mitochondria of motor axon terminals, as well as in rough endoplasmic reticulum and Golgi complex of motor neuron perikarya, but not in sensory neurons or Purkinje cells. The reason for the selective vulnerability of motor neurons is not clearly defined, but a prominent possibility is the physiological absence in motor neurons of the calcium-binding proteins calbindin-D28k and parvalbumin. These studies emphasize the central role of increased intracellular calcium in motor neuron cell death in sporadic ALS, and the role of autoimmunity in triggering such increases.

Apoptotic cell death of a hybrid motoneuron cell line induced by immunoglobulins from patients with amyotrophic lateral sclerosis.

Apoptotic cell death has recently been implicated in diseases involving nonproliferating, terminally differentiated cells such as neurons. Previous experiments have documented that immunoglobulins from patients with amyotrophic lateral sclerosis (ALS) can kill motoneuron-neuroblastoma hybrid cells [ventral spinal cord 4.1 (VSC 4.1)] by a calcium-dependent process. Here, we studied the mechanism of ALS IgG-induced cell death. In the presence of ALS IgG the VSC 4.1 cells undergo cell shrinkage and membrane blebbing, which are morphological features of apoptotic cell death. The damaged cells can be identified by in situ end labeling of nicked DNA and biochemically show laddering on agarose gel electrophoresis. This ALS IgG-triggered process is prevented by cycloheximide, aurintricarboxylic acid, and zinc sulfate. These data demonstrate that immunoglobulins from patients with ALS are able to induce apoptosis in motoneuron hybrid cells and provide a potential mechanism for motoneuron degeneration in human ALS.

Extrinsic modulation of medial septal cell discharges by the ascending brainstem hippocampal synchronizing pathway.

Single cells or simultaneously recorded cell pairs in the medial septum (MS) vertical limb of the diagonal band of Broca (vDBB) in the urethane-anesthetized rat were initially classified as either theta-on or non-related according to the system of Colom and Bland (1987, Brain Res 422:277-286). Subgroups of these cells were then studied under various test conditions that included electrical stimulation of the nucleus pontis oralis (PO) or posterior hypothalamus (PH), microinfusion of carbachol into the PO or PH, and the microinfusion of atropine sulfate or procaine hydrochloride into the PH. Electrical stimulation of either the PO or PH induced theta (theta) activity in the hippocampal formation (HPC), and electrical stimulation of the PO resulted in a simultaneous increase in the discharge rate of all MS/vDBB theta-on cells tested, compared to the rates recorded during HPC large-amplitude irregular activity (LIA). Five of the MS/vDBB theta-on cells were tested consecutively with electrical stimulation of the PO and PH, and were shown to be activated in a similar manner in either condition. Microinfusion of carbachol into either the PO or PH resulted in the induction of HPC theta field activity and the simultaneous intense activation of all MS/vDBB theta-on cells tested. Following the microinfusion of either atropine sulfate or procaine into the PH, electrical stimulation of the PO failed to induce HPC theta field activity or the concomitant rhythmic discharges of all MS/vDBB phasic theta-on cells tested. Microinfusing procaine into the PH also abolished the coupling between all MS/vDBB cell pairs during HPC theta field activity including that between two cell pairs that were coupled during HPC LIA. The data support the following conclusions: 1) The brainstem HPC synchronizing pathway originating in the pons region ascends to the medial septum via the midline posterior hypothalamic region; 2) the present results taken together with previous work suggest that a major component of the ascending synchronizing pathway, up to and including the hippocampal formation, is cholinergic, cholinoceptive, or both, and the receptors involved are primarily muscarinic; 3) the midline posterior hypothalamic region is an important source of inputs to the medial septum and their major contribution is to provide frequency-coded inputs to the MS/vDBB for relay into the hippocampal formation.

The midline posterior hypothalamic region comprises a critical part of the ascending brainstem hippocampal synchronizing pathway.

Electrical stimulation and microinfusion techniques were utilized in acute experiments on urethane-anesthetized rats in order to evaluate the hypothesis that the posterior hypothalamic and supramammillary nuclei comprise a critical part of the ascending brainstem pathway for producing synchronous hippocampal formation (HPC) field activity (theta). Given confirmation of this hypothesis a second objective was to determine the nature of the contribution made by this midline posterior hypothalamic region (PH) to the frequency and amplitude components of HPC theta field activity. The cholinergic nature of the ascending pathway was also examined. Reversible inactivation of the PH was achieved by microinfusion of the local anesthetic procaine hydrochloride. The efficacy of and recovery from procaine inactivation of the PH was quantitatively analyzed either by electrical stimulation of the nucleus pontis oralis (PO) (two experiments) or the PH (four experiments). The results are summarized under the following three headings: 1) The first is the effect of procaine inactivation of the PH on HPC theta elicited caudal to, at the level of, or rostral to the PH. All HPC theta induced caudal to the PH (spontaneous theta, tail pinch-induced theta, and theta produced by electrical stimulation of the PO) was totally abolished for a minimum 10-min period. HPC theta induced rostral to the PH by the intrahippocampal infusion of carbachol was unaffected, while HPC theta induced by infusions of carbachol into either the medial septum (MS) or PH was reduced in amplitude with no effect on frequency. 2) Next are comparisons of pre- and post-PH procaine trials of electrical stimulation of the PO and PH. In all experiments, regardless of the anatomical locus or technique used to induce HPC theta, pre- and post-PH procaine comparisons of the PO and PH stimulation trials revealed that frequency modulation of HPC theta recovered significantly more slowly than amplitude. 3) Last is the effect of electrical stimulation of the PO and PH on HPC theta induced by carbachol infusions at the level of the HPC, MS, or PH. In all experiments, electrical stimulation of both the PO and PH, at appropriate intensities, resulted in increasing HPC theta frequencies above the frequency induced by the infusion of carbachol into the HPC, MS, and PH. In addition, the post-carbachol HPC theta frequencies induced by electrical stimulation were significantly higher than those produced in the pre-carbachol conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Spontaneous interictal-like activity originates in multiple areas of the CA2-CA3 region of hippocampal slices.

1. The sites of origin of spontaneous interictal-like epileptiform activity in hippocampal slices from guinea pig, mouse, and rat were determined. A multisite fast optical recording technique using voltage-sensitive dyes and an array of 100 photodiodes was employed. The use of a low-magnification objective lens allowed the visualization of almost the entire transverse hippocampal slice. Three in vitro models of epilepsy were employed, utilizing different manipulations of the bath perfusion medium to induce epileptiform activity: 1) raising the external potassium (K+) concentration, 2) adding the potassium channel blocker 4-aminopyridine (4-AP), and 3) adding antagonists of gamma-aminobutyric acid-A (GABAA) receptors (bicuculline and picrotoxin, BIC-PTX). 2. Spontaneous epileptiform discharges were detected in each subfield of cornu ammonis (CA) but not in the dentate gyrus (DG) of each studied species. Preliminary experiments confirmed that interictal-like epileptiform activity originated in the CA2-CA3 region. Ictal-like activity was never observed in our experiments. 3. In the guinea pig, when GABAA antagonists were employed, the site of origin of spontaneous epileptiform discharges was consistently located in the CA2-CA3a region. When high K+ or 4-AP was used, this region was the most frequent site of origin. Subsequent epileptiform discharges with similar sites of origin occasionally invaded different areas of the CA2-CA3 region, revealing a variable area of occupance of epileptiform discharges. 4. In the mouse and rat, the site of origin of spontaneous discharges was invariably located in the CA3b-CA3c region independent of the epilepsy model. 5. In both the guinea pig and rat, when the CA2-CA3a region was surgically separated from the CA3b-CA3c region, independent discharges were observed in both regions. Areas that could generate discharges only under certain epileptogenic conditions were found in these species (potential sites of origin). Two independent sites of origin with different propagation patterns and area of occupance were occasionally observed within the CA2-CA3a region. 6. In the guinea pig, such lesions demonstrated that both regions can independently generate epileptiform discharges at different frequencies. When high K+ or 4-AP was employed, epileptiform activity was observed in both regions. Although BIC-PTX only generated discharges in the CA2-CA3a region, a subsequent increase in K+ induced additional discharges in the CA3b-CA3c region, revealing a potential site of origin. 7. In rat hippocampal slices with such lesions, spontaneous epileptiform discharges were observed in both CA2-CA3a and CA3b-CA3c region when 4-AP was employed.(ABSTRACT TRUNCATED AT 400 WORDS)