Effect of FK960, a putative cognitive enhancer, on synaptic transmission in CA1 neurons of rat hippocampus.

The action of FK960 [N-(4-acetyl-1-piperazinyl)-p-fluorobenzamide monohydrate], a novel cognitive enhancer, on excitatory synaptic transmission in the hippocampus was investigated. Excitatory postsynaptic potentials (EPSPs) and currents (EPSCs) were recorded intracellularly from CA1 neurons in rat hippocampus using the "blind patch" variant of whole-cell recording. FK960 (100 nM) significantly increased the amplitude of the EPSP, which was unchanged when changeover was made to control artificial cerebrospinal fluid (aCSF). FK960 had no significant action on membrane potential, input resistance, or the early GABAergic inhibitory postsynaptic current. The decay phase of the excitatory postsynaptic current was not significantly altered by exposure to FK960, indicating that the properties of desensitization and/or deactivation were unchanged and suggesting that the action of FK960 was unlikely to be the result of changes in the properties of the postsynaptic (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptors. The quantal content of the EPSP (1/CV(2)) increased after exposure to FK960 but not to control aCSF. Methyllycaconitine or alpha-bungarotoxin blocked the modulatory action of FK960 on the EPSP, and the finding that these alpha 7-nicotinic acetylcholine receptor (alpha 7nAChR) antagonists were effective raises the possibility that FK960 up-regulates the contribution of acetylcholine to synaptic efficacy in the hippocampus. It is concluded that FK960 increases the quantal release of glutamate from Schaffer collateral-commissural nerve terminals in area CA1 of the hippocampus either by changing the ambient level of acetylcholine or by positively modulating the activity of alpha 7nAChRs located on glutamatergic nerve terminals.

Only 'de novo' long-term depression (LTD) in the rat hippocampus in vitro is blocked by the same low concentration of FK506 that blocks LTD in the visual cortex.

It has been proposed that the long-term depression (LTD) seen following low frequency stimulation (LFS) in the rat hippocampus involves calcineurin. We have tested this by examining the effect of FK506, a macrolide which blocks calcineurin at nanomolar concentrations, on synaptic transmission in the rat hippocampal slice at a concentration of 1 microM which has been shown to block LTD in the visual cortex. The effect of FK506 on long-term potentiation (LTP) and spontaneous transmitter release was also studied. The magnitude of LTD induced by LFS was 16.7 +/- 2.4% in control which was not significantly different from the 22.3 +/- 3.0% seen in the same preparations after exposure to FK506 for 25-30 min. In contrast the magnitude of LTD induced 'de novo' in preparations exposed to FK506 was significantly reduced. FK506 had no significant effect on LTP, miniature EPSP frequency, miniature EPSP amplitude, resting membrane potential or input resistance. These results, therefore, support the hypothesis that calcineurin is involved in 'de novo' LTD but it appears that an event is triggered by LFS whereby FK506-insensitive LTD can subsequently be activated by a second episode of LFS.

Neurochemical and electrophysiological studies on FR115427, a novel non-competitive NMDA receptor antagonist.

The pharmacological profile of FR115427 has been examined using ligand binding and electrophysiological techniques. Binding of [3H]dizocilpine in the presence of L-glutamate was inhibited by the (+) isomers of dizocilpine and FR115427. The corresponding (-) isomers were less active, and stereoselectivity was particularly marked in the case of FR115427. In contrast to dizocilpine, the affinity of FR115427 for [3H]dizocilpine binding sites was little affected by addition of either L-glutamate and/or glycine. In a cortical wedge preparation, FR115427 inhibited N-methyl-D-aspartate (NMDA)-induced responses in a non-competitive, use-dependent manner. Intracellularly recorded excitatory synaptic responses in hippocampal neurones were only partially inhibited by FR115427 thereby confirming a selective effect on the NMDA-mediated component of neuronal excitation induced by the endogenous neurotransmitter. The data suggest that FR115427 is a non-competitive, use-dependent NMDA receptor antagonist with more pronounced stereoselectivity and less marked use dependence than dizocilpine.

An intracellular study of the action of NAN-190 on neurons in the dorsal raphe nucleus of the rat.

Intracellular recordings have been made from neurons in the dorsal raphe (DR) nucleus of the rat to determine the mechanism of action of the arylpiperazine compound NAN-190. Application of NAN-190 (50 microM) alone most frequently caused a hyperpolarization accompanied by a fall in RM and reduced the response to 5-HT and 8-OH-DPAT. After pretreatment of the preparation with the 5-HT-uptake blocker citalopram (10 microM) NAN-190 exerted an excitatory effect. It is concluded that NAN-190 is a partial agonist in the DR nucleus.

An intracellular study of grafted and in situ preoptic area neurones in brain slices from normal and hypogonadal mice.

1. Intracellular recordings have been obtained from forty-one preoptic area (POA) neurones at times up to 14 months after they were grafted into the third ventricle of the mouse. Thirty-one neurones were in grafts from hypogonadal (hpg) mice in which a reversal of the hypogonadism was seen (responders), six were in grafts from hpg mice in which no such reversal occurred (non-responders) and four were in grafts from normal mice. 2. The grafted neurones had a mean resting potential (Em) of -57 mV, a mean apparent input resistance (Rm) of 136 M omega and a mean membrane time constant (tau m) of 7.7 ms. The slopes of the current-voltage (I-V) relations were linear. Approximately a quarter of neurones in responders fired action potentials spontaneously either singly or in bursts. Such activity could underlie the release of gonadotrophin hormone-releasing hormone (GnRH) which is known to occur from such grafts. 3. Two types of response were seen when these neurones were depolarized to firing threshold from Em, in one group a single action potential was discharged; in the other group one or more action potentials arising from a transient, slowly rising and falling depolarization (low-threshold response, LTR) was recorded. Some cells in the former category exhibited a LTR when depolarized from a potential more negative than Em. 4. The commonest response to stimulation of the median eminence in responders was an EPSP either alone or in combination with an IPSP. Antidromic action potentials were seen in four neurones and in two of these cells excitatory synaptic inputs could be demonstrated when the host hypothalamus adjacent to the graft was stimulated. It is suggested that these responses may represent activation of an afferent input from the host to neurones in the graft. 5. The morphology of neurones in POA grafts was determined by intrasomatic injection of horseradish peroxidase (HRP). A variety of profiles were seen and although some neurones extended over distances of up to 635 microns and branched extensively only one appeared to enter the host tissue at the ventrolateral edge of the graft. 6. A comparison was made between grafted POA neurones and cells in the medial preoptic area (MPOA), a region which constituted a significant component of the grafted tissue. No significant difference was noted between neurones in the graft and neurones in the MPOA in terms of their passive membrane properties. With regard to the active properties MPOA neurones could also be classified according to whether or not a LTR was elicited when the neurone was depolarized from Em. The major difference between the grafted neurones and those in the MPOA lay in the proportion of cells which exhibited a LTR under such conditions, being significantly greater in the latter group.

Intrinsic reflexes underlying peristalsis in the small intestine of the domestic fowl.

Peristalsis in the chicken small intestine was studied using either a modified Trendelenburg method or a technique in which changes in circular muscle activity were recorded in response to application of a localized radial distension. A localized radial distension had no effect on either the resting tension or the spontaneous activity of the circular muscle on the oral side of distension. On the aboral side of the distension a transient contraction was recorded in the ileum and jejunum after a mean delay of 2.74 s at 37 degrees C. In about a third of the preparations a tonic contraction was also present which persisted for as long as distension was maintained. The transient contraction was blocked by hyoscine (0.6-2.3 microM) and hexamethonium (275 microM); whereas the tonic contraction persisted in the presence of hyoscine. Both types of contraction were blocked by tetrodotoxin (0.31 microM). No such responses were recorded in the duodenum. The descending excitatory reflex responses were followed in all preparations by a fall in the amplitude and frequency of spontaneous contractions and in a few preparations by a concomitant fall in the tone of the circular muscle lasting for up to 3 min. This inhibitory component of the descending reflex was not blocked by guanethidine (3-10 microM). The transient contraction, which originated most frequently at the site of distension, always propagated aborally at a mean speed of 14.2 mm s-1. Surgical interruption of the longitudinal muscle and myenteric plexus effectively blocked the transmission of the excitatory and inhibitory components of the descending reflex past the site of the lesion. In the modified Trendelenburg apparatus raising the intraluminal pressure elicited peristalsis in the isolated ileum. Peristaltic contractions never started at the most oral end of the preparation but appeared instead at any other point on the ileum. This resulted in several contractions contributing to each emptying cycle. Peristalsis was blocked by tetrodotoxin (0.31 microM). These results are discussed in the terms of the organization of the descending reflex. It is suggested that within the enteric nervous system of the ileum and jejunum of the chicken, there are cholinergic and non-cholinergic excitatory neurones and non-adrenergic inhibitory neurones. The results of this study demonstrate that neurogenic peristalsis in the avian small intestine does not conform to the 'law of the intestine' as originally postulated by Bayliss & Starling (1899).

The unmyelinated fibre spectrum of the main trunk and side branches of the intestinal nerve in the chicken (Gallus gallus var. domesticus).

An electron microscopy study was undertaken to determine the numbers and diameters of unmyelinated axons in the main trunk and side branches of the intestinal nerve at the level of the small intestine. Three techniques were used to determine axon diameter. These were: length of the least chord (D), diameter determined from cross sectional area (DA) and diameter determined from perimeter (DP). A measure of the degree of circularity of the axons was also made. On average, the nerve trunk contained 4729 unmyelinated axons and these outnumbered myelinated axons by 78:1. The mean number of unmyelinated axons in the side branches was 463. Myelinated axons were not seen in the side branches. When a test for circularity was applied to the data it was found that larger axons tended to be less circular than smaller ones and, because of these differences in the degree of circularity, DA and more particularly D were subject to a variable inaccuracy. The mean values for DA were 0.70 micron for the nerve trunk and 0.57 micron for the side branch; the mean values for DP were 0.78 and 0.66 microns respectively. The peak conduction velocity at 40 degrees C of unmyelinated axons in the nerve trunk was determined from the single (C-fibre) deflection in the compound action potential. The following relationship was found, peak conduction velocity CV = DA0.59 = DP0.85.

A study of pre- and postganglionic fibres in the intestinal nerve (Remark's nerve) of the chicken.

The mean peak CV's of two electrophysiologically defined groups of fibres in the intestinal nerve of the chicken have been determined. One group of fibres is constituted by the processes of enteric cholinergic neurones which project along the side branches of the intestinal nerve and synapse within the nerve trunk. These preganglionic fibres have a mean peak CV (at 40 degrees C) of 0.31 m x s-1. The other group is made up of fibres of postganglionic neurones which project orally along the nerve trunk. The results suggest that some postganglionic neurones project only as far as the next ganglion whilst others project beyond the next two ganglia for distances greater than 5 mm. The postganglionic fibres have a mean peak CV (at 40 degrees C) of 0.71 m X s-1. These figures demonstrate that both pre- and postganglionic fibres are unmyelinated. The temperature coefficient (Q10) for the CV of unmyelinated fibres in the intestinal nerve was 1.57.

The conduction velocity, number, and diameter of unmyelinated fibers in Remak's nerve.

Remak's nerve in the chicken was examined ultrastructurally and electrophysiologically to determine the characteristics of fibers in the nerve trunk. The ration of unmyelinated fibers to myelinated ones was 111:1. The mean number of unmyelinated fibers was 3555 plus/minus 232 (SEM, n=5) and they had a mean diameter of 0.502 plus/minus 0.034 (SEM) micron. The compound action potential consisted almost entirely of a large diphasic waveform which had a mean peak conduction velocity of 0.62 plus/minus 0.031 (SEM, n=5) m.s-1 at 37 degrees C.

Influence of vagotomy in domestic fowls on feeding activity, food passage, digestibility and satiety effects of two peptides.

The effects of bilateral vagotomy at the level of the proventriculus, in immature female fowls (VAG), on body weight, feeding activity parameters, rate of food passage, digestibility, and satiety effects of bombesin (BBS) and cholecystokinin octapeptide (CCK8), were compared with those in sham-operated controls (SHAM). SHAM birds gained weight at a greater rate postoperatively than before their operation, whereas VAG birds did not. Daily food intake did not change significantly as a result of the operation with either SHAM or VAG birds, and the only effect on feeding activity parameters was on the length of intermeal intervals, which increased in VAG birds. Rate of passage of the layers' mash diet was slower, and its apparent digestibility lower, in VAG than in SHAM birds. Short-term suppression of food intake, following intravenous injections of BBS (10 micrograms/kg) or CCK8 (10 micrograms/kg), did not differ between SHAM and VAG birds. The different postoperative weight gains may have been a consequence of different weights of food digested, and the difference in interval length was probably due to the different rates of food passage. The results of these experiments indicate that efferent information affecting rate of passage and digestibility travels via the vagus, but that afferent information concerned with initiation and termination of meals, and with satiety effects of BBS and CCK8, does not. Instead, such afferent information may travel via the intestinal nerve, which is unique to birds.

Evidence that enteric cholinergic neurones project orally in the intestinal nerve of the chicken.

The intestinal nerve of the chicken, which runs almost the whole length of the small and large intestine, was studied in vitro, using suction electrodes for stimulation and recording. Lengths of nerve with side branches intact in the region of the ileum and rectum were used. The spike discharges that were evoked by supramaximal stimulation of a side branch were recorded at oral and aboral ends of the main nerve trunk. Exposure of the preparation to hexamethonium (550 microM) reversibly abolished or greatly reduced the amplitude of the largest component of the evoked oral response in eighteen experiments, but was without effect on the evoked aboral response in ten experiments. No evidence of a nicotinic cholinergic component to the evoked aboral response was obtained at distances up to 12.5 mm along the nerve. There was also no evidence for a projection of post-ganglionic neurones along the stimulated side branch. The mean conduction velocity of the preganglionic nerve fibres was 0.25 +/- 0.02 m. s-1 while that for the post-ganglionic nerve fibres was 0.66 +/- 0.11 m.s-1. These results suggest that both groups of nerve fibres are non-myelinated. 8-16 d after side branch transection, stimulation of the side branch evoked no oral response in five experiments and a hexamethonium-insensitive response in seven others. In a further four experiments a hexamethonium-sensitive component was seen, but it constituted only a small part of the evoked oral response. It is postulated that transection results in the degeneration of cholinergic nerves in the intestinal nerve. The results suggest that enteric cholinergic nerves form part of a pathway which projects orally along the ileal and rectal segments of the intestinal nerve.

Peristalsis and antiperistalsis in the chicken caecum are myogenic.

Peristalsis in the chicken caecum was investigated by recording the responses of isolated segments of the caecum to either fluid distension using a modified Trendelenburg apparatus or by monitoring circular muscle activity following localized radial distension of adjacent areas. Raising the intraluminal pressure did not initiate peristalsis except in the presence of tetrodotoxin, local anaesthetics and high concentrations of phentolamine. The effect of these drugs was reversible. Localized distension generally produced either a small relaxation of the circular muscle or no response at all on both sides of the site of distension. After exposure to tetrodotoxin one, or more (usually several), rhythmic propogating contractions were initiated at the point of distension. These results suggest that intrinsic inhibitory neurones are present in the caecum and may be triggered by distention. The possible role of these inhibitory neurones in the emptying and filling of the caecae is considered.

Slow intracellular potentials in AH-neurons of the myenteric plexus evoked by repetitive activation of synaptic inputs.

In after-hyperpolarization-neurons of guinea-pig myenteric plexus, several types of slow responses to repetitive activation of synaptic inputs were recorded. There were at least two types of slow excitatory depolarizing potential, both associated with an increased input resistance, but in one there was an abolition or marked attenuation of the amplitude of the slow after-hyperpolarization of the neuron, whereas in the other type the slow after-hyperpolarization was not significantly affected. Slow depolarizing and hyperpolarizing synaptic potentials associated with a decrease input resistance were also recorded in some neurons following transmural stimulation. The neural processes responsible for these slow potentials in after-hyperpolarization-neurons showed no preferential projection within the myenteric plexus.

Morphological studies of electrophysiologically-identified myenteric plexus neurons of the guinea-pig ileum.

The morphology of neurons in the myenteric plexus of the guinea-pig ileum has been studied by means of the intracellular application of Procion Yellow. Sixty-six electrophysiologically-unidentified cells showed a great variety of soma shapes and number of processes, the vast majority of the longer of which were circumferentially-orientated. The electrophysiological properties of an additional 47 neurons were ascertained; 29 were neurons with a slow after-hyperpolarization (AH-neurons), 14 showed fast excitatory synaptic potentials (S-neurons) and 4 were of neither category. Twenty-two of the AH-neurons had a smooth soma outline and, on average, each had 5 processes, of which the great majority of long processes were circumferentially-orientated and intraganglionic. The projection ratios of oral:circumferential:aboral processes were 6:61:9. Branching was a prominent feature of the processes. In contrast, a large soma with many broad, short processes was a feature of 8 of 14 S-neurons studied. The average number of processes was 8.6 per cell and relatively more of them were aborally-directed, giving projection ratios of 2:21:7. There was, however, such a variation and overlap in the morphology of AH- and S-neurons that it was not possible to achieve a simple, reliable classification. It is concluded that many neuronal processes may be intraganglionic and that longitudinal ones are mainly aboral. From the varied morphological characteristics of AH-neurons, it is unlikely that these neurons subserve a single function in the plexus. For the same reasons S-neurons may fulfil different physiological roles.

Sub-miniature junction potentials in excitatory neuromuscular synapses of the locust.

By recording miniature excitatory junction potentials (mejps) intracellularly at two points from a multiterminally innervated muscle fibre it is possible to select mejps whose amplitudes are not substantially affected by electrotonic decay. Many amplitude histograms of such selected mejps from untreated locust jumping muscle show a bimodal distribution with a high proportion of small-amplitude mejps (sub-mejps). Most amplitudes of excitatory junction potentials (ejps) resulting from the release of a single transmitter quantum correspond to the large-mode mejps. Tetanic nerve stimulation, in high [Mg2+]o without Ca2+, greatly reduces the proportion of sub-mejps. It is concluded that there are two modes of spontaneous transmitter release from the motor nerve endings.

Slow synaptic potentials in AH-type myenteric plexus neurons.

Two types of slow depolarization were recorded in AH-type guinea pig myenteric plexus neurons when the myenteric plexus-longitudinal muscle preparation was stimulated transmurally with external electrodes. One depolarization was associated with a fall and the other with a rise in membrane resistance, the latter type (slow EPSP) being encountered about six times more commonly than the former. In some instances both types of potential were recorded in the same AH neuron. When this occurred the amplitude and duration of the slow EPSP was attenuated if it was timed to occur at about the same time as the other slow synaptic potential.

Transmitter release from normal and degenerating locust motor nerve terminals.

1. An analysis has been made of spontaneous and evoked transmitter release from terminals of 'fast' excitatory motor axons on locust muscle fibres using intra- and extracellular recording together with a Ca-electrode technique for activating transmitter release from single nerve terminals on multiterminally innervated muscle fibres. 2. Spontaneous intracellular miniature excitatory junction potentials (m.e.j.p.s), recorded at active spots on these muscle fibres, occurred non-randomly with frequent bursts of m.e.j.p.s. 3. M.e.j.p.s of subnormal amplitude were also seen but contributed only a small fraction of the minature discharge. 4. The amplitude distribution of intracellularly recorded excitatory junction potentials (e.j.p.s) evoked during ionophoretic application of Ca onto single nerve terminals was adequately predicted by Poisson statistics. 5. During the course of nerve terminal degeneration m.e.j.p.s of subnormal amplitude became more frequent and eventually formed the major part of the miniature discharge. Transmitter quanta responsible for 'small' m.e.j.p.s did not contribute to evoked release either at normal or degenerating terminals. Evoked transmitter release from degenerating axon terminals before excitation-secretion coupling failure conformed to Poisson statistics. 6. It is concluded that more than one release mechanism operates on the transmitter pool or pools in locust motor nerve terminals.