Expression and cellular function of vSNARE proteins in brain astrocytes.

Gray matter protoplasmic astrocytes, a major type of glial cell in the mammalian brain, extend thin processes ensheathing neuronal synaptic terminals. Albeit electrically silent, astrocytes respond to neuronal activity with Ca(2+) signals that trigger the release of gliotransmitters, such as glutamate, d-serine, and ATP, which modulate synaptic transmission. It has been suggested that the astrocytic processes, together with neuronal pre- and post-synaptic elements, constitute a tripartite synapse, and that astrocytes actively regulate information processing. Astrocytic vesicles expressing VAMP2 and VAMP3 vesicular SNARE (vSNARE) proteins have been suggested to be a key feature of the tripartite synapse and mediate gliotransmitter release through Ca(2+)-regulated exocytosis. However, the concept of exocytotic release of gliotransmitters by astrocytes has been challenged. Here we review studies investigating the expression profile of VAMP2 and VAMP3 vSNARE proteins in rodent astrocytes, and the functional implication of VAMP2/VAMP3 vesicles in astrocyte signaling. We also discuss our recent data suggesting that astrocytic VAMP3 vesicles regulate the trafficking of glutamate transporters at the plasma membrane and glutamate uptake. A better understanding of the functional consequences of the astrocytic vSNARE vesicles on glutamate signaling, neuronal excitability and plasticity, will require the development of new strategies to selectively interrogate the astrocytic vesicles trafficking in vivo.

Expression of a functional hyperpolarization-activated current (Ih) in the mouse nucleus reticularis thalami.

The GABAergic neurons of the nucleus reticularis thalami (nRT) express the type 2 hyperpolarization-activated cAMP-sensitive (HCN2) subunit mRNA, but surprisingly, they were reported to lack the hyperpolarization-activated (Ih) current carried by this subunit. Using the voltage-clamp recordings in the thalamocortical slice preparation of the newborn and juvenile mice (P6-P23), we demonstrate that, in the presence of 1 mM barium (Ba2+), the nRT neurons express a slow hyperpolarization-activated inward current, suggesting that the Ih is present but masked in control conditions by K+ leak currents. We investigate the identity of the hyperpolarization-activated current in the nRT by studying its physiological and pharmacological profile in presence of Ba2+. We show that it has voltage- and time-dependent properties typical of the Ih, that it is blocked by cesium and ZD7288, two blockers of the Ih, and that it is carried both by the K+ and Na+ ions. We could also alter the gating characteristics of the hyperpolarization-activated current in the nRT by adding a nonhydrolysable analogue of cAMP to the pipette solution. Finally, using the current-clamp recording, we showed that blocking the hyperpolarization-activated current induced an hyperpolarization correlated with an increase of the R(in) of the nRT neurons. In conclusion, our results demonstrate that the nRT neurons express the Ih with slow kinetics similar to those described for the homomeric HCN2 channels, and we show that the Ih of the nRT contributes to the excitability of the nRT neurons in normal conditions.

Intermediate zone cells express calcium-permeable AMPA receptors and establish close contact with growing axons.

Recent studies have shown that cells in the intermediate zone (IZ) of the embryonic neocortex originate in the basal telencephalon and migrate tangentially in the cortical wall (;; ). We had previously observed growing cortical axons closely apposed to calbindin-positive, tangentially oriented cells in the IZ (), and it has been shown that neurites in the IZ express a glutamate transporter (). To test if glutamate released by corticofugal growth cones could influence the tangential IZ cells, we characterized the glutamate receptors expressed by IZ cells using patch-clamp techniques, histochemical labeling, and immunostaining on slices of embryonic mice forebrain. We show that tangential IZ cells express inwardly rectifying kainate responses, but not NMDA responses, and accumulate cobalt after AMPA receptor activation. We conclude that IZ cells express calcium-permeable AMPA receptors. This property correlates with our observation that the GluR2 subunit is not expressed in the IZ. AMPA receptors are activated by a millimolar concentration of glutamate. To know whether this high level of glutamate could occur at the surface of IZ cells, we examined contacts made by corticofugal growth cones and calbindin-positive IZ cells using electron microscopy. We show vesicle-containing neurites tightly apposed to calbindin-positive IZ cells over remarkably long length. This suggests that glutamate released by growing corticofugal axons could reach high concentrations close to AMPA receptors of tangential IZ cells and efficiently activate them to control the intracellular calcium in embryonic IZ cells.

Altering the concentration of GABA in the synaptic cleft potentiates miniature IPSCs in rat occipital cortex.

We have tested the effect of dextran (40 kDa, 5%) on miniature IPSCs (mIPSCs) recorded in layer V cortical pyramidal cells. This compound increases the amplitude of mIPSCs at room and physiological temperatures by 15%, leaving their duration unaffected at room temperature and slightly increased at physiological temperature. The amplitude increase is attributable to an increase in the number of receptors bound by GABA during synaptic transmission, as shown by the occlusion between the effects of dextran and zolpidem on mIPSC amplitude at room temperature. As dextran presumably enhances the concentration and dwell time of GABA in the synaptic cleft, these results demonstrate that the postsynaptic GABAA receptors are not saturated at room and physiological temperatures.

Effect of zolpidem on miniature IPSCs and occupancy of postsynaptic GABAA receptors in central synapses.

GABAA-mediated miniature IPSCs (mIPSCs) were recorded from layer V pyramidal neurons of the visual cortex using whole-cell patch-clamp recording in rat brain slices. At room temperature, the benzodiazepine site agonist zolpidem enhanced both the amplitude (to 138 +/- 26% of control value at 10 microM) and the duration (163 +/- 14%) of mIPSCs. The enhancement of mIPSC amplitude was not caused by an increase of the single-channel conductance of the postsynaptic receptors, as determined by peak-scaled non-stationary fluctuation analysis of mIPSCs. The effect of zolpidem on fast, synaptic-like (1 msec duration) applications of GABA to outside-out patches was also investigated. The EC50 for fast GABA applications was 310 microM. In patches, zolpidem enhanced the amplitude of currents elicited by subsaturating GABA applications (100-300 microM) but not by saturating applications (10 mM). The increase of mIPSC amplitude by zolpidem provides evidence that the GABAA receptors are not saturated during miniature synaptic transmission in the recorded cells. By comparing the facilitation induced by 1 microM zolpidem on outside-out patches and mIPSCs, we estimated the concentration of GABA seen by the postsynaptic GABAA receptors to be approximately 300 microM after single vesicle release. We have estimated a similar degree of receptor occupancy at room and physiological temperature. However, at 35 degreesC, zolpidem did not enhance the amplitude of mIPSCs or of subsaturating GABA applications on patches, implying that, in these neurons, zolpidem cannot be used to probe the degree of receptor occupancy at physiological temperature.

Molecular and physiological diversity of cortical nonpyramidal cells.

The physiological and molecular features of nonpyramidal cells were investigated in acute slices of sensory-motor cortex using whole-cell recordings combined with single-cell RT-PCR to detect simultaneously the mRNAs of three calcium binding proteins (calbindin D28k, parvalbumin, and calretinin) and four neuropeptides (neuropeptide Y, vasoactive intestinal polypeptide, somatostatin, and cholecystokinin). In the 97 neurons analyzed, all expressed mRNAs of at least one calcium binding protein, and the majority (n = 73) contained mRNAs of at least one neuropeptide. Three groups of nonpyramidal cells were defined according to their firing pattern. (1) Fast spiking cells (n = 34) displayed tonic discharges of fast action potentials with no accommodation. They expressed parvalbumin (n = 30) and/or calbindin (n = 19) mRNAs, and half of them also contained transcripts of at least one of the four neuropeptides. (2) Regular spiking nonpyramidal cells (n = 48) displayed a firing behavior characterized by a marked accommodation and presented a large diversity of expression patterns of the seven biochemical markers. (3) Finally, a small population of vertically oriented bipolar cells, termed irregular spiking cells (n = 15), fired bursts of action potentials at an irregular frequency. They consistently co-expressed calretinin and vasoactive intestinal polypeptide. Additional investigations of these cells showed that they also co-expressed glutamic acid decarboxylase and choline acetyl transferase. Our results indicate that neocortical nonpyramidal neurons display a large diversity in their firing properties and biochemical patterns of co-expression and that both characteristics could be correlated to define discrete subpopulations.

Expression of GABA(A) receptor subunit mRNAs by layer V pyramidal cells of the rat primary visual cortex.

The expression of the GABA(A) receptor subunit mRNAs by layer V pyramidal neurons of the primary visual cortex and cerebellar Purkinje cells was analysed by single-cell reverse transcription of the mRNAs and amplification of the resulting cDNAs by the polymerase chain reaction. Neurons were identified by infrared videomicroscopy, and GABA(A)-mediated miniature inhibitory postsynaptic currents were recorded. In Purkinje cells, alpha1, beta2, beta3, gamma2S and gamma2L subunit mRNAs were detected within a single cell. In layer V pyramidal cells, a total of ten GABA(A) receptor subunit mRNAs could be detected, with a mean of seven subunit mRNAs per cell, suggesting GABA(A) receptor heterogeneity within a single pyramidal cell.

Correlation between kinetics and RNA splicing of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors in neocortical neurons.

In the cortex fast excitatory synaptic currents onto excitatory pyramidal neurons and inhibitory nonpyramidal neurons are mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors exhibiting cell-type-specific differences in their kinetic properties. AMPA receptors consist of four subunits (GluR1-4), each existing as two splice variants, flip and flop, which critically affect the desensitization properties of receptors expressed in heterologous systems. Using single cell reverse transcription PCR to analyze the mRNA of AMPA receptor subunits expressed in layers I-III neocortical neurons, we find that 90% of the GluR1-4 in nonpyramidal neurons are flop variants, whereas 92% of the GluR1-4 in pyramidal neurons are flip variants. We also find that nonpyramidal neurons predominantly express GluR1 mRNA (GluR1/GluR1-4 = 59%), whereas pyramidal neurons contain mainly GluR2 mRNA (GluR2/GluR1-4 = 59%). However, the neuron-type-specific splicing is exhibited by all four AMPA receptor subunits. We suggest that the predominance of the flop variants contributes to the faster and more extensive desensitization in nonpyramidal neurons, compared to pyramidal cells where flip variants are dominant. Alternative splicing of AMPA receptors may play an important role in regulating synaptic function in a cell-type-specific manner, without changing permeation properties.

Diversity of glutamate receptors in neocortical neurons: implications for synaptic plasticity.

The biochemical and functional characteristics of the AMPA subtype of the glutamate receptors expressed by pyramidal and non-pyramidal neurons of the neocortex have been studied in acute slices by means of single-cell RT-PCR and fast applications of glutamate on outside-out patches. Our results suggest that the predominant expression of the flop splice variants of the GluR1-4 AMPA subunits contributes to the faster desensitization of these receptors in non-pyramidal neurons compared to pyramidal cells where flip variants of GluR1-4 are dominant. Alternative splicing of AMPA receptors may therefore play an important role in regulating synaptic function in a cell-type specific manner.

Serotonin facilitates GABAergic transmission in the CA1 region of rat hippocampus in vitro.

1. The effect of serotonin on inhibitory synaptic transmission was examined in forty-one CA1 pyramidal neurones using intracellular voltage recordings in vitro. 2. Serotonin (20-50 microM) increased the synaptic noise of most (85%) neurones loaded with chloride (n = 33). The duration of this effect was enhanced with increasing concentrations of serotonin and was fully reversible within 5 min. When serotonin was applied at short intervals (less than 10 min), fading of the response was observed. 3. The effect of serotonin on synaptic noise persisted in the presence of the glutamate NMDA and non-NMDA antagonists, APV (100 microM) and CNQX (10 microM), but it was blocked (n = 5) by a GABAA antagonist, bicuculline (10 microM). 4. The increase in inhibitory synaptic events resulted from an enhanced frequency of unitary IPSPs from 4.6 +/- 3.8 Hz in control to 17.2 +/- 12.5 Hz (n = 5) in serotonin, especially of large events. Serotonin caused no change in the amplitude and frequency of miniature synaptic events recorded in the presence of TTX (n = 5). The mean amplitude of unitary inhibitory postsynaptic potentials (IPSPs) increased from 1.37 +/- 0.35 mV in control to 3.67 +/- 1.38 mV in serotonin. The coefficient of variation of unitary IPSPs increased from 0.40 +/- 0.11 in control to 0.74 +/- 0.23 in serotonin when quantal size appeared unchanged. 5. The 5-HT3 agonist 2-methyl-serotonin (52 microM, n = 4) partially mimicked the effect of serotonin, increasing the inhibitory noise without affecting the pyramidal neurone conductance. The serotonin-induced facilitation of unitary IPSPs was blocked by the 5-HT3 antagonists ICS 205-930 (1-90 nM, n = 3) and metoclopramide (30 microM, n = 1). 6. These results suggest that serotonin directly excites GABAergic interneurones acting on a 5-HT3 receptor and consequently increasing the frequency of inhibitory synaptic events recorded in CA1 pyramidal cells.

Characteristics of miniature inhibitory postsynaptic currents in CA1 pyramidal neurones of rat hippocampus.

1. Recordings were made in vitro from chloride-loaded CA1 rat hippocampal pyramidal neurones in the presence of tetrodotoxin (TTX) to examine miniature inhibitory postsynaptic currents (IPSCs). 2. Most spontaneous synaptic events recorded before TTX was applied, and all events that were resolved in the presence of TTX, were blocked by the GABAA receptor antagonist bicuculline. 3. At 25 degrees C, averaged miniature IPSCs had time to peak of about 3 ms and in most cases decayed with a single time constant close to 25 ms. 4. With a driving force for chloride ions between 70 and 80 mV, the mean miniature IPSC amplitude was 19.6-27.9 pA, yielding a conductance of 258-326 pS. The mean amplitude of unitary IPSCs recorded before TTX was applied was in the range of 31-73 pA. 5. When intervals between miniature IPSCs were compared with an exponential distribution, there was an excess of events at intervals shorter than 5 ms. Some individual events appeared to represent the nearly simultaneous release of two inhibitory quanta. 6. Miniature IPSC amplitude distributions were better fitted with the sum of two Gaussians than with one Gaussian. The variance in amplitude of a single quantal event exceeded that of the baseline noise. 7. Comparison of the conductance changes corresponding to the first Gaussian distribution with single GABA channel data suggests that one inhibitory quantum opens twelve to twenty chloride channels and that GABA molecules bind once to a postsynaptic receptor.

The protein kinase C inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) disinhibits CA1 pyramidal cells in rat hippocampal slices.

1. The effects of the protein kinase C (PKC) inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) on evoked synaptic potentials were investigated in the CA1 region of rat hippocampal slices by use of extracellular and intracellular recording techniques. 2. Extracellular recordings showed that superfusion with H-7 (10-100 microM) increased the amplitude of the population spike and the initial slope of the dendritic field e.p.s.p. H-7 also produced the appearance of multiple population spikes in the somatic region and in the dendritic field e.p.s.p. 3. H-7 (30 microM) induced the disappearance of intracellularly recorded inhibitory potentials elicited by orthodromic stimulation of CA1 pyramidal cells. At this concentration H-7 had no effect on resting membrane potential, input membrane resistance, and spike threshold. In voltage-clamped neurones H-7 blocked the antidromically evoked inhibitory currents and the spontaneous miniature inhibitory currents. 4. The hyperpolarizing effect of bath applied gamma-aminobutyric acid (GABA, 500 microM) or isoguvacine (30 microM) was not affected by 30 microM H-7. 5. Neither the PKC activity regulator sphingosine (10-40 microM) nor the H-7 analogue N-(2-guanidinoethyl)-5-isoquinolinesulphonamide (HA-1004, 20-50 microM) which is devoid of activity on PKC at these concentrations, affected the extracellularly recorded dendritic field e.p.s.p. or population spike. 6. It is concluded that the disinhibitory effect produced by H-7 is due to the block of a H-7-sensitive PKC which is involved in the spontaneous and evoked release of GABA.

Inhibitory action of serotonin in CA1 hippocampal neurons in vitro.

The ionic mechanism of the inhibitory effect of serotonin was investigated in vitro in the CA1 region of the rat hippocampus by extra- and intracellular recordings. Local or bath applications of serotonin induced a long-lasting reduction of extracellularly recorded synaptic potentials and orthodromic population spikes without affecting the afferent volley or the antidromic population spike. Serotonin can also reduce the frequency of occurrence of spontaneous excitatory and inhibitory postsynaptic potentials without any reduction of input resistance of the pyramidal neuron. During the response to serotonin, the conductance increase evoked by GABA, the inhibitory neurotransmitter, was not changed. A direct postsynaptic effect of serotonin was demonstrated: local or bath applications of serotonin induced a tetrodotoxin-resistant hyperpolarization and conductance increase. The conductance change was not reduced by manual clamp of the neurons to the control resting membrane potential; therefore, a possible involvement of the sodium-potassium electrogenic pump is unlikely. When neurons were loaded with chloride, serotonin could still induce a hyperpolarization with an apparent reversal more negative than the resting membrane potential. When neurons were loaded with caesium, the hyperpolarization and the conductance increase evoked by serotonin were blocked. It is therefore concluded that serotonin increases potassium permeability. Similar effects were induced by a 5-HT1A ligand. The slow after hyperpolarization was reduced by serotonin; the calcium spike was reduced at the same time. In caesium loaded neurons, the spike duration was not modified by serotonin. In the presence of extracellular caesium (4-5 mM), the serotonin-induced hyperpolarization and the conductance change were blocked, but the effect of serotonin on calcium spikes persisted. Tetraethylammonium (5-10 mM) or 4-aminopyridine (0.5 mM) had no effect on the response to serotonin. These data indicate that serotonin has a postsynaptic inhibitory action by an activating potassium conductance. The possibility of a regulation of calcium currents is discussed. The possible role of serotonin on intrinsic synaptic transmission is also discussed.

Septohippocampal disinhibition.

Intracellular recordings from CA1 and CA2/3 neurons in rats under urethane anesthesia revealed the following effects of medial septal stimulation (10 pulse trains at approximately 100 Hz): (1) in most cases only minimal signs of any synaptic potential; (2) a marked and prolonged (200-500 ms) depression of on-going inhibitory postsynaptic potentials (IPSPs), particularly evident when IPSPs were reversed by Cl- injection; (3) a corresponding increase in input resistance: (4) depolarization when recording with non-Cl(-)-containing electrodes; (5) a predominant hyperpolarization when recording with Cl(-)-containing electrodes; and (6) a marked reduction of the variability of resistance and voltage data. These observations indicate that septal stimulation can strongly depress tonic inhibition in the hippocampus. Septal trains also tended to weaken IPSPs evoked in pyramidal cells by fimbrial stimulation, reducing conductance increase during IPSPs by an average of 42% (S.D. +/- 24.3). Septal inputs to the hippocampal CA1 and CA2/3 regions appear to have a major disinhibitory function.

Measurements of intracellular free Ca2+ in mammalian central neurons.

Neutral carrier-containing Ca2+-selective microelectrodes were used to record the cytoplasmic free Ca2+ concentration [( Ca2+]i) in spinal cells in cats and in hippocampal cells of rats (in situ). The mean [Ca2+]i in motoneurons was close to 1 microM. Antidromic or direct stimulation for 30 s at 10 Hz increased [Ca2+]i by a mean of 90 nM. Such a small increase in [Ca2+]i and its slow decay (with a mean half-time of 23 (SD +/- 14.5) s) indicate very effective intracellular sequestration of Ca2+. Orthodromic stimulation consistently evoked smaller increases in [Ca2+]i. A much larger rise of interneuronal [Ca2+]i was evoked by stimulation of dorsal roots: by contrast intra-axonal recording (in motor or sensory fibres) failed to reveal any increase in [Ca2+]i in response to stimulation at 100 Hz. In the hippocampus, presumably because of poorer recording conditions, resting values of [Ca2+]i were higher (mean 8.5 microM). Repetitive stimulation of the fimbria--commissure at 5-20 Hz for 30 Hz, had variable effects on [Ca2+]i. Very large increases (to greater than 200 microM) were elicited repeatedly in some cells, either near the end of the tetanic stimulation or after a 20-30 s delay. Such major increases, which were associated with population cell discharges in bursts, may be related to long-term changes in hippocampal neuronal properties that are evoked by tetanic stimulation. Both in the spinal cord and the hippocampus, probable intraglial recordings showed relatively high mean levels of [Ca2+]i (about 30 microM).

Changes in free calcium ion concentration recorded inside hippocampal pyramidal cells in situ.

In rats under urethane or pentobarbitone anesthesia, Ca2+ -sensitive microelectrodes were inserted into CA3 and CA1 hippocampal cells. In 23 neurons with a mean resting membrane potential (Vm) of -56.9 mV, the Ca potential (VCa) fell below Vm by an average of -22.1 mV (S.D. +/- 19.1 mV), indicating a mean intracellular free Ca2+ concentration ([Ca]i) of 9.7 microM (S.D. 14.9 microM). In spite of their better and more stable Vm (mean -67.1 mV), unresponsive cells (probably neuroglia) had a higher and more variable [Ca]i (mean 37.0 +/- 51.2 microM). In 21 of the neurons, repetitive stimulation of the fimbria--at 5-20 Hz for 30s, which is sufficient to elicit bursts of population spikes--evoked substantial increases in [Ca]i: the mean increase observed during or just after 29 such tetani was +27.1 +/- 54.5 microM. Typically [Ca]i reached a peak near the end of the tetanus and then decayed with a half-time of 5-10 s, though not necessarily to the initial level. In 7 cells, a large increase in [Ca] (mean +239 +/- 367 microM) appeared as a late event, 20-30 s after the end of the tetanus. In 5 cells, [Ca]i could thus be raised transiently to 10(-4) M or higher. All these increases in [Ca]i are far greater than can be evoked by tetanic activation in spinal motoneurons; their possible significance for long term potentiation or cell necrosis in the hippocampus is discussed.

Chemical modulation of ephaptic activation of CA3 hippocampal pyramids.

In rats under urethane anaesthesia, antidromic population spikes were evoked in CA3 pyramidal layer by fimbrial/commissural stimulation at a very low frequency (approximately 0.5 Hz). Submaximal population spikes--between 20 and 90% of maximum--were enhanced by 8-38% by applications of acetylcholine and bicuculline, or by medial septal stimulation. Noradrenaline had a less pronounced and regular facilitatory action, whereas gamma-aminobutyrate and glutamate only depressed population spikes. Maximal enhancement by acetylcholine or bicuculline was observed when the antidromic population spike was initially at 38-53% of maximum amplitude. A simple explanation of these results is that acetylcholine and bicuculline, by raising their excitability, facilitate the excitation of non-invaded pyramidal cells by antidromic field potentials. They are fully in keeping with previous intracellular observations on ephaptic interactions between CA3 neurons, and provide a further illustration, in situ, of the importance of increased excitability and disinhibition--whether caused by drugs or synaptic action--in promoting synchronized excitation by ephaptic currents.

Modulation of inhibition in the hippocampus in vivo.

The nature and mechanisms of septohippocampal transmission have been elucidated by taking advantage of an in situ preparation in experiments with Sprague-Dawley rats under urethane. Both extracellular field potentials and intracellular recordings were made in CA1-3 regions of the hippocampus; and the hippocampal commissure and medial septum stimulated to evoke synaptic activity. Using muscarinic and nicotinic agonists and antagonists it was shown that both acetylcholine and medial septal activity can increase the excitability of pyramidal cells, mainly through muscarinic receptors. The effect of septal stimulation was enhanced by local application of physostigmine and reduced by intraventricular injections of hemicholinium. It was also shown that acetylcholine, when applied in the stratum pyramidale, can reduce the voltage and conductance changes observed during evoked inhibitory postsynaptic potentials (IPSP) without affecting the action of gamma-aminobutyric acid on membrane conductance and voltage. It is therefore proposed that acetylcholine can reduce evoked IPSPs through presynaptic inhibition. Evidence is also presented that medial septal stimulation can reduce the efficacy of evoked IPSPs. These observations provide further support for the existence of a cholinergic septohippocampal pathway.

Facilitation of hippocampal CA3 pyramidal cell firing by electrical fields generated antidromically.

In experiments on rats under urethane anaesthesia--in which the fimbria and hippocampal commissure had been cut previously to eliminate orthodromic inputs--the negative antidromic population spike evoked in CA3 by fimbrial stimulation was measured inside and outside 73 neurons in the stratum pyramidale. Subtraction of the extracellular from the intracellular records showed that on the average 39.2% (S.E. 1.93) of the extracellular population spikes appeared as a positive, depolarizing transmembrane potential. Similar measurements in the dendritic zone of CA3, where the extracellular antidromic population spike is positive, revealed a smaller and hyperpolarizing transmembrane potential, whereas presumed neuroglia showed no consistent transmembrane potential in either direction. Further tests demonstrated clear facilitation of individual pyramidal cell firing, synchronous with the antidromic population spike. These observations are consistent with the possibility that, owing to the unusually close packing and regular alignment of the pyramidal neurons, electrical field interactions in CA3 tend to promote synchronized mass discharges.

Hemicholinium impairs septo-hippocampal facilitatory action.

Intraventricular injections of hemicholinium-3 led to a sharp reduction in hippocampal acetylcholine content (by 79% on the average). This was associated with the following changes in population spikes evoked in area CA1 by commissural stimulation: (1) a tendency to progressive increase, over 1-2 hours; (2) in a few cases (3 out of 12), a striking depression of the normal strong facilitation produced by brief tetanic stimulation of the medial septum (typically 10 pulses at 50-100 Hz); (3) a much more consistent tendency towards fading of the septal facilitatory effect during repeated applications of such brief septal tetani (in 10 cases out of 12); as well as, (4) diminished facilitation by sustained, lower frequency septal tetanic stimulation (20-50 Hz). The reduced efficiency of septal action--especially during repetitive stimulation--was not accompanied by a consistent reduction of the facilitation produced by local applications of acetylcholine; it is, therefore, best explained by the diminished availability of acetylcholine, and so provides further evidence that septo-hippocampal facilitation is mediated by a cholinergic mechanism.