Graded synaptic transmission between identified spiking neurons

Graded synaptic transmission between spiking motoneurons of the pyloric group was studied in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus. Intracellular microelectrodes were placed in the cell bodies of both pre- and postsynaptic neurons. Graded synaptic transmission was found between all tested cell pairs that were known to display spike-evoked synaptic transmission, including PD to LP, PD to PE, PD to PL, PL to LP, and LP to PD. Graded synaptic transmission was effective below the threshold for spikes. Thus, it was possible to study the influence of graded synaptic transmission in normally active ganglia without blockage of spikes by tetrodotoxin. PD and LP neurons that were known to produce spike-evoked inhibitory postsynaptic potentials (IPSPs) were also capable of producing inhibitory effects on postsynaptic cells below the threshold for spikes. When tetrodotoxin (TTX) was used to eliminate both spikes and endogenous membrane oscillations, depolarization of presynaptic neurons produced hyperpolarization of postsynaptic cells. The presynaptic response to a current step usually showed a small early peak and a maintained, slightly lower plateau. The postsynaptic response had a delay, then a rise to a pronounced peak, and a roughly exponential decline to a maintained plateau. There was a presynaptic voltage threshold for any postsynaptic response; beyond the threshold, both pre- and postsynaptic peak and plateau responses increased with increasing current. PD neurons normally are depolarized beyond their release threshold in tetrodotoxin and, thus, released transmitter tonically for the many-hour duration of these experiments. Chemical, tonic synaptic transmission, here called graded synaptic transmission, was demonstrated by the presence of the following criteria: 1) reversal in sign of the postsynaptic response, 2) synaptic delay, 3) reversal potential, 4) postsynaptic conductance increase, 5) graded and reversible block by reduction of external Ca2+, and 6) specific graded block of the LP-to-PD synapse without effect on the PD-to-LP synapse by less than 10 microM picrotoxin added to the bathing medium.     

Analysis of synaptic transmission at single identified boutons on rat spinal neurons in culture

The spatial organization of receptor channels has a major influence on the speed and possible plasticity of synaptic signal transmission. We have studied glutamatergic synapses on neurons in organotypic cultures of rat spinal cord. In order to avoid the problems related to the analysis of currents of unknown origin within a neuron, we chose to examine the functional properties of single identified synapses. Iontophoretic mapping of the cell surface revealed hot spots of high glutamate sensitivity coincident with presynaptic boutons stained with the dye FM 1–43. Local application of KCl to these sites caused bursts of synaptic release. Hot spots typically consisted of 330 receptors with an average single-channel conductance of 8.3 pS. Evoked synaptic currents involved only about 40–50 receptors and nevertheless showed characteristics of saturation. This suggests that glutamate receptor clusters at sites of presynaptic terminals are organized into well separated subclusters opposite release sites.This award-winning article is published as received and has not been subjected to the normal peer review process     

Dopamine regulates synaptic transmission mediated by cholinergic neurons of the rat retina

The purpose of this study was to investigate the effect of dopamine on the function of synapses formed by cholinergic neurons derived from the rat retina. We used an experimental culture system in which rat striated muscle cells served as postsynaptic targets for cholinergic neurons of the retina. This culture system permitted the physiological monitoring of acetylcholine release at synapses formed by retinal neurons. We found that dopamine could facilitate evoked transmission at retina-muscle synapses. This facilitation by dopamine was reversible and could be blocked by haloperidol, a dopamine receptor antagonist. The adenosine 3':5'-phosphate analogue, 8-bromoadenosine 3':5'-phosphate, mimicked the facilitating effect of dopamine. In addition, dopamine elevated markedly the levels of adenosine 3':5'-phosphate in cultures of rat retinal cells. The results suggest that dopamine can regulate transmission through retinal neurons. Our findings support the hypothesis that a dopamine-induced facilitation of stimulus-evoked transmission involves the activation of dopamine receptors and the intracellular accumulation of adenosine 3':5'-phosphate.     

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.     

Phenotypic changes of morphologically identified guinea-pig myenteric neurons following intestinal inflammation

We investigated the responses of morphologically identified myenteric neurons of the guinea-pig ileum to inflammation that was induced by the intraluminal injection of trinitrobenzene sulphonate, 6 or 7 days previously. Electrophysiological properties were examined with intracellular microelectrodes using in vitro preparations from the inflamed or control ileum. The neurons were injected with marker dyes during recording and later they were recovered for morphological examination. A proportion of neurons with Dogiel type I morphology, 45% (32/71), from the inflamed ileum had a changed phenotype. These neurons exhibited an action potential with a tetrodotoxin-resistant component, and a prolonged after-hyperpolarizing potential followed the action potential. Of the other 39 Dogiel type I neurons, no changes were observed in 36 and 3 had increased excitability. The afterhyperpolarizing potential (AHP) in Dogiel type I neurons was blocked by the intermediate conductance, Ca²⁺-dependent K⁺ channel blocker TRAM-34. Neurons which showed these phenotypic changes had anally directed axonal projections. Neither a tetrodotoxin-resistant action potential nor an AHP was seen in Dogiel type I neurons from control preparations. Dogiel type II neurons retained their distinguishing AH phenotype, including an inflection on the falling phase of the action potential, an AHP and, in over 90% of neurons, an absence of fast excitatory transmission. However, they became hyperexcitable and exhibited anodal break action potentials, which, unlike control Dogiel type II neurons, were not all blocked by the h current ( I _h) antagonist Cs⁺. It is concluded that inflammation selectively affects different classes of myenteric neurons and causes specific changes in their electrophysiological properties.     

Long-term actions of BDNF on inhibitory synaptic transmission in identified neurons of the rat substantia gelatinosa

Peripheral nerve injury promotes the release of brain-derived neurotrophic factor (BDNF) from spinal microglial cells and primary afferent terminals. This induces an increase in dorsal horn excitability that contributes to "central sensitization" and to the onset of neuropathic pain. Although it is accepted that impairment of GABAergic and/or glycinergic inhibition contributes to this process, certain lines of evidence suggest that GABA release in the dorsal horn may increase after nerve injury. To resolve these contradictory findings, we exposed rat spinal cord neurons in defined-medium organotypic culture to 200 ng/ml BDNF for 6 days to mimic the change in spinal BDNF levels that accompanies peripheral nerve injury. Morphological and electrophysiological criteria and glutamic acid decarboxylase (GAD) immunohistochemistry were used to distinguish putative inhibitory tonic-islet-central neurons from putative excitatory delay-radial neurons. Whole cell recording in the presence of 1 μM tetrodotoxin showed that BDNF increased the amplitude of GABAergic and glycinergic miniature inhibitory postsynaptic currents (mIPSCs) in both cell types. It also increased the amplitude and frequency of spontaneous, action potential-dependent IPSCs (sIPSCs) in putative excitatory neurons. By contrast, BDNF reduced sIPSC amplitude in inhibitory neurons but frequency was unchanged. This increase in inhibitory drive to excitatory neurons and decreased inhibitory drive to inhibitory neurons seems inconsistent with the observation that BDNF increases overall dorsal horn excitability. One of several explanations for this discrepancy is that the action of BDNF in the substantia gelatinosa is dominated by previously documented increases in excitatory synaptic transmission rather than by impediment of inhibitory transmission.     

Neurokinin-1 and -3 receptor blockade inhibits slow excitatory synaptic transmission in myenteric neurons and reveals slow inhibitory input

Recent studies have shown that tachykinins mediate slow synaptic transmission to myenteric AH (afterhyperpolarising) neurons via neurokinin-3 receptors (NK(3)R). This study investigated a similar role for neurokinin-1 receptors (NK(1)R) and compared the effect of selective receptor antagonists on non-cholinergic slow excitatory post-synaptic potentials (EPSPs) recorded in myenteric AH neurons of the guinea-pig ileum. Slow EPSPs evoked by electrical stimulation of circumferentially oriented presynaptic nerves were mimicked by application of senktide, an NK(3)R agonist. [Sar(9),Met(O(2))(11)]-substance P, an NK(1)R agonist, depolarised a smaller number of neurons. SR142801, a selective NK(3)R antagonist (100 nM), inhibited slow EPSPs and responses to senktide, but had no effect on depolarisations evoked by forskolin, an activator of adenylate cyclase. SR140333, a selective NK(1)R antagonist, inhibited slow EPSPs in a subset of neurons and blocked responses to [Sar(9),Met(O(2))(11)]-substance P, but not to senktide or forskolin. Slow EPSPs that were predominantly mediated by NK(1)R had significantly shorter latencies than those due to activation of NK(3)R. After blockade of slow EPSPs, slow hyperpolarizing responses to presynaptic nerve stimulation were revealed in one-third of neurons. These events, which were associated with a decrease in input resistance and blocked by tetrodotoxin, were equated with slow inhibitory postsynaptic potentials. They were abolished by the 5-hydroxytryptamine(1A) receptor antagonist 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]-piperazine (NAN-190), but unaffected by phentolamine, an alpha-adrenoceptor antagonist. In conclusion, these results provide the first direct evidence that NK(1)R mediate some slow excitatory synaptic input to myenteric AH neurons, and suggest that NK(1)R and NK(3)R activate distinct signal transduction pathways. These results also demonstrate that slow inhibitory synaptic transmission, which may be mediated by 5-hydroxytryptamine, is more prevalent in the myenteric plexus than previously indicated.     

Effects of adenosine on gabaergic synaptic inputs to identified ventrolateral preoptic neurons

The ventrolateral preoptic nucleus (VLPO) is a key regulator of behavioral state that promotes sleep by directly inhibiting brain regions that maintain wakefulness. Subarachnoid administration of adenosine (AD) or AD agonists promotes sleep and induces expression of Fos protein in VLPO neurons. Therefore, activation of VLPO neurons may contribute to the somnogenic actions of AD. To define the mechanism through which AD activates VLPO neurons, we prepared hypothalamic slices from 9 to 12-day-old rat pups and recorded from 43 neurons in the galaninergic VLPO cluster; nine neurons contained galanin mRNA by post hoc in situ hybridization. Bath application of AD (20 microM) to seven of these neurons had no direct effect but caused a significant decrease in the frequency of spontaneous miniature inhibitory postsynaptic currents in the presence of tetrodotoxin, indicating a presynaptic site of action. We conclude that AD-mediated disinhibition increases the excitability of VLPO neurons thus contributing to the somnogenic properties of AD.     

Comparison of the effects of neurokinin-3 receptor blockade on two forms of slow synaptic transmission in myenteric AH neurons

AH neurons are intrinsic sensory neurons of the intestine that exhibit two types of slow synaptic event: slow excitatory postsynaptic potentials which increase their excitability for about 2-4 min, and sustained slow postsynaptic excitation which can persist for several hours, and may be involved in long-term changes in the sensitivity of the intestine to sensory stimuli. The effects of the neurokinin-3 tachykinin receptor antagonist, SR142801, on these two types of synaptic event in AH neurons of the myenteric ganglia of guinea-pig small intestine were compared. Slow excitatory postsynaptic potentials were evoked by stimulation of synaptic inputs at 10-20 Hz for 1s, and sustained slow postsynaptic excitation was evoked by stimulation of inputs at 1Hz for 4 min. SR142801 (1microM) reduced the amplitude of the slow excitatory postsynaptic potential to 26% of control, and also reduced the increase in input resistance and the extent of anode break excitation associated with the slow excitatory postsynaptic potential. In contrast, SR142801 did not reduce the increase in excitability, the increase in input resistance or the depolarisation that occur during the sustained slow postsynaptic excitation. SR142801 did not change the resting membrane potential or the resting input resistance.We conclude that tachykinins, acting through neurokinin-3 receptors, are involved in the generation of the slow excitatory postsynaptic potential, but not in the sustained slow postsynaptic excitation, and that the release of transmitters from synaptic inputs to AH neurons is frequency coded.     

Excitatory synaptic potentials due to activation of neurons with short projections in the myenteric plexus

Intracellular microelectrodes have been used to examine the effects, on excitatory inputs to myenteric nerve cells, of lesions of intrinsic pathways in the myenteric plexus of the guinea-pig small intestine. The lesions consisted of circumferential cuts (myotomies) which severed the external musculature to the depth of the submucosa and thus interrupted pathways in the myenteric plexus. Sufficient time was allowed between creating the lesions and recording from the neurons for the endings of severed neurites to degenerate and this was confirmed histochemically by examining the distribution of varicose fibres with 5-hydroxytryptamine immunoreactivity in myenteric ganglia from which recordings were made. Two types of excitatory input, eliciting fast and slow excitatory post-synaptic potentials, respectively, were demonstrable in response to focal stimulation of nerves in the ganglia from which recordings were made. There were no differences in the proportions of neurons in which fast or slow excitatory synaptic potentials were evoked in unoperated preparations (controls), in islands 1.5-4 mm wide between myotomies, or within 1 mm on the oral or anal sides of myotomies. Possible differences in the amplitudes, durations at half amplitude, and threshold numbers of stimuli for initiation of slow excitatory synaptic potentials were analyzed. The only significant differences were found when data from control and oral areas were pooled and compared with combined data from island and anal areas (this assessed differences that could arise from severing nerve fibres running from oral to anal).(ABSTRACT TRUNCATED AT 250 WORDS)     

Naloxone-induced depolarization and synaptic activation of myenteric neurons in morphine-dependent guinea pig ileum

To investigate the cellular basis of opiate dependence, intracellular microelectrodes were used to record from both electrophysiologically defined classes of neurons (S and AH) in myenteric plexus longitudinal muscle preparations from morphine pretreated guinea pigs. These preparations responded to naloxone with the characteristic contraction of the longitudinal smooth muscle, indicative of morphine dependence. Depolarization in response to naloxone was observed in 42% of S neurons, but there were no consistent changes in input resistance. In some cells the depolarization was reduced or abolished after blockade of synaptic transmission, suggesting that it was due in part to the release of an excitatory transmitter producing a slow depolarization in the impaled neuron. Synaptic activation of S neurons during withdrawal was further indicated by the observation that fast postsynaptic potentials appeared after abrupt displacement of morphine from its receptors by naloxone. Morphine withdrawal, therefore, involves both the final motor neurons and interneurons. During naloxone-induced withdrawal, 25% of S neurons discharged action potentials. In contrast, no action potentials were discharged in AH neurons. Furthermore, naloxone did not alter the resting membrane potential, input resistance, soma action potential configuration, or slow hyperpolarization following a soma spike in AH neurons. The specificity of the withdrawal response for S neurons and the relatively small proportion of neurons involved suggests that morphine withdrawal occurs in quite specific neuronal circuits in the myenteric plexus.     

Isoflurane-induced impairment of synaptic transmission in hippocampal neurons

Summary The effects of anaesthetic applications of isoflurane were studied using intracellular recording techniques in 82 CA₁ neurons of in vitro hippocampal slice preparations (guinea pigs). Various parameters of their excitabilities such as membrane electrical properties, action potentials evoked by intracellular current pulse injections and spike afterhyperpolari-zations, as well as synaptic potentials evoked by electrical stimulation of stratum radiatum, were determined during bath perfusion of clinical concentrations of isoflurane which were measured with ¹⁹fluorine-nuclear magnetic resonance techniques. The vaporizer settings of 1–4% isoflurane corresponded to concentrations of 100 M to 500 M. Isoflurane applications did not produce consistent effects on the resting potentials or passive membrane properties. However, when spike-evoked synaptic activity was blocked by tetrodotoxin, isoflurane application induced a hyperpolarization (3–5 mV) without greatly affecting input conductance and the slopes of current-voltage relations. The threshold, amplitude and duration of single or multiple spikes evoked by current injections also were not greatly altered by isoflurane applications. However, marked reductions were observed in the amplitudes of the long-lasting hyperpolarizations following an evoked train of a constant number (4 or 5) of spikes. The amplitudes of excitatory postsynaptic potentials evoked by electrical stimulation of stratum radiatum were diminished markedly during isoflurane applications; these effects, like those on the afterhyperpolarizations, were closely dependent on the dose and duration of the application. Low doses (<1%) of isoflurane reduced the amplitudes of inhibitory postsynaptic potentials whereas higher doses (1–4%) increased their amplitudes and durations. The effects on afterhyperpolarizations and synaptic potentials could not be attributed to anaesthetic related changes in the resting potentials of the neurons. The investigations provide evidence for presynaptic, as well as postsynaptic, sites of isoflurane actions in hippocampal neurons which presumably may involve direct alterations in intracellular Ca²⁺-disposition or inward Ca²⁺-currents.     

Significance of slow synaptic potentials for transmission of excitation in guinea-pig myenteric plexus

Intracellular recordings were made from neurones in myenteric ganglia of the guinea-pig ileum in vitro. Synaptic potentials were evoked by electrically stimulating presynaptic fibres as they entered the ganglion, using a small focal electrode. Slow synaptic depolarizations (excitatory postsynaptic potentials) were evoked in most myenteric neurones of both types. A single stimulus was more likely to evoke a slow excitatory postsynaptic potential in cells with nicotinic synaptic input (S cells; 50%) than in cells with long-lasting after-hyperpolarizations following the soma action potential (AH cells; 20%). Two pulses often evoked a slow excitatory postsynaptic potential in AH cells when one pulse was ineffective. The optimally effective time between the pulses was about 100 ms. Ten pulses resulted in slow excitatory postsynaptic potentials even when delivered at frequencies as low as 0.5 Hz. For the same frequency of presynaptic stimulation, the duration of the slow excitatory postsynaptic potential was greater in AH cells than in S cells and the amplitude of the slow excitatory postsynaptic potential was slightly greater in S than AH cells. Spontaneous depolarizations were observed which had time-courses and amplitudes similar to the evoked slow excitatory postsynaptic potential. They were not blocked by tetrodotoxin or atropine. The calcium-dependent after-hyperpolarization which follows one or more action potentials in AH cells was reduced or even abolished during the slow excitatory postsynaptic potential. Presynaptic nerve stimulation at intensities lower than those required to cause a slow excitatory postsynaptic potential caused a reduction in the calcium dependent after-hyperpolarization. It is concluded that the slow excitatory postsynaptic potential is generated by an intracellular intermediate process which is sensitive to the intracellular calcium concentration. The results suggest that the postsynaptic action of the synaptic transmitter is to interfere with the intracellular process which couples the entry of calcium to the increase in potassium conductance.     

Projections of chemically identified myenteric neurons of the small and large intestine of the mouse

The projections of different subpopulations of myenteric neurons in the mouse small and large intestine were examined by combining immunohistological techniques with myotomy and myectomy operations. The myotomies were used to examine the polarity of neurons projecting within the myenteric plexus and showed that neurons containing immunoreactivity for nitric oxide synthase (NOS), vasoactive intestinal peptide (VIP), calbindin and 5-HT projected anally, while neurons with substance P (SP)-immunoreactivity projected orally, in both the small and large intestine. Neurons containing neuropeptide Y (NPY)- and calretinin-immunoreactivity projected locally. In the large intestine, GABA-immunoreactive neurons projected both orally and anally, with more axons tending to project anally. Myectomy operations revealed that circular muscle motor neurons containing NOS/VIP/±NPY and calretinin neurons projected anally both in the small and large intestine, while SP-immunoreactive circular muscle motor neurons projected orally. In the large intestine, GABA-IR circular muscle motor neurons projected both orally and anally. This study showed that although some neurons, such as the NOS/VP inhibitory motor neurons and interneurons, SP excitatory motor neurons and 5-HT interneurons had similar projections to those in other species, the projections of other chemical classes of neurons in the mouse intestine differed from those reported in other species.     

Immunohistochemical identification of cholinergic neurons in the myenteric plexus of guinea-pig small intestine.

It is well established that acetylcholine is a neurotransmitter at several distinct sites in the mammalian enteric nervous system. However, identification of the cholinergic neurons has not been possible due to an inability to selectively label enteric cholinergic neurons. In the present study an immunohistochemical method has been developed to localize choline acetyltransferase, the synthetic enzyme for acetylcholine, in order that cholinergic neurons can be visualized. The morphology, neurochemical coding and projections of cholinergic neurons in the guinea-pig small intestine were determined using double-labelling immunohistochemistry. These experiments have revealed that many myenteric neurons are cholinergic and that they can be distinguished by their specific combinations of immunoreactivity for neurochemicals such as calretinin, neurofilament protein triplet, substance P, enkephalin, somatostatin, 5-hydroxytryptamine, vasoactive intestinal peptide and calbindin. On the basis of their previously described projections, functional roles could be attributed to each of these populations. The identified cholinergic neurons are: motorneurons to the longitudinal muscle (choline acetyltransferase/calretinin); motorneurons to the circular muscle (choline acetyltransferase/neurofilament triplet protein/substance P, choline acetyltransferase/substance P and choline acetyltransferase alone); orally directed interneurons in the myenteric plexus (choline acetyltransferase/calretinin/enkephalin); anally directed interneurons in the myenteric plexus (choline acetyltransferase/somatostatin, choline acetyltransferase/5-hydroxytryptamine, choline acetyltransferase/vasoactive intestinal peptide); secretomotor neurons to the mucosa (choline acetyltransferase/somatostatin); and sensory neurons mediating myenteric reflexes (choline acetyltransferase/calbindin). This information provides a unique opportunity to identify functionally distinct populations of cholinergic neurons and will be of value in the interpretation of physiological and pharmacological studies of enteric neuronal circuitry.     

Leptin modulates fast synaptic transmission in dog pancreatic ganglia

The aim of this study was to determine whether leptin modulates neuronal activity in intrapancreatic ganglion neurons. Intracellular recordings were made in dog pancreatic neurons. Recombinant mouse leptin (313 nM) was added by superfusion. When leptin was present, fast EPSPs which were subthreshold in normal Krebs solution reached threshold for firing action potentials. However, leptin had no significant (P > 0.05, n = 18) effect on either the resting membrane potential or on membrane input resistance. To determine whether leptin increased the postsynaptic sensitivity to acetylcholine, the response was tested by pressure ejection of acetylcholine. Acetylcholine evoked a 9.4+/-2.2 mV (mean +/- SEM, n = 5) depolarization in normal Krebs solution. In the presence of leptin, the response was not significantly different (9.6+/-2.4 mV, P > 0.05). The results suggest that leptin modulates fast synaptic transmission in pancreatic ganglion neurons by acting on presynaptic nerve terminals.