Separation of electrophysiologically distinct neuronal populations in the rat hippocampus for neuropharmacological testing under in vivo conditions.

Microiontophoresis combined with extracellular spike recording is an excellent method for investigating local neuropharmacological effects under in vivo conditions. However, its application has recently become relatively rare in neuroscience research. Now, we aimed to revisit microiontophoresis and demonstrate that it provides valuable data about the pharmacophysiology of neurons and local neuronal networks, in vivo. Extracellular recordings were performed through the central recording channel of multibarrel carbon-fiber microelectrodes in the CA1 pyramidal layer of the hippocampus of anesthetized rats, while N-methyl-D-aspartate (NMDA) was locally administrated by means of microiontophoresis through the surrounding micropipettes of the microelectrode. Various separation procedures were used to distinguish putative pyramidal cells and interneurons. Quality of separation was verified by electrophysiological parameters. After the delivery of NMDA in the vicinity of the examined neurons, firing rate of putative pyramidal cells was increased with a significantly higher grade then that of putative interneurons. The present results in line with previous data indicate that pyramidal cells are more responsive to pharmacological manipulation through NMDA receptors, also confirming the reliability of the separation of different types of neurons in in vivo microiontophoretic experiments.

Pentapeptides derived from Abeta 1-42 protect neurons from the modulatory effect of Abeta fibrils--an in vitro and in vivo electrophysiological study.

Short fragments and fragment analogues of beta-amyloid 1-42 peptide (Abeta1-42) display a protective effect against Abeta-mediated neurotoxicity. After consideration of our earlier results with in vitro bioassay of synthetic Abeta-recognition peptides and toxic fibrillar amyloids, five pentapeptides were selected as putative neuroprotective agents: Phe-Arg-His-Asp-Ser amide (Abeta4-8) and Gly-Arg-His-Asp-Ser amide (an analogue of Abeta4-8), Leu-Pro-Tyr-Phe-Asp amide (an analogue of Abeta17-21), Arg-Ile-Ile-Gly-Leu amide (an analogue of Abeta30-34), and Arg-Val-Val-Ile-Ala amide (an analogue of Abeta38-42). In vitro electrophysiological experiments on rat brain slices demonstrated that four of these peptides counteracted with the field excitatory postsynaptic potential-attenuating effect of Abeta1-42; only Arg-Val-Val-Ile-Ala amide proved inactive. In in vivo experiments using extracellular single-unit recordings combined with iontophoresis, all these pentapeptides except Arg-Val-Val-Ile-Ala amide protected neurons from the NMDA response-enhancing effect of Abeta1-42 in the hippocampal CA1 region. These results suggest that Abeta recognition sequences may serve as leads for the design of novel neuroprotective compounds.

The modulatory effect of estrogen on the neuronal activity in the barrel cortex of the rat. An electrophysiological study.

In acute experiments, the effects of iontophoretically applied 17 beta-estradiol hemisuccinate on the activity of the primary somatosensory cortical neurons were studied in ovariectomized rats by extracellular single-unit recording. 17 beta-Estradiol increased both the spontaneous and the vibrissa deflection-evoked responses, with an average latency of 24 min. It is suggested that this relatively long latency of the 17 beta-estradiol effect is based not so much on membrane mechanisms as on genomic mechanisms.

Midbrain periaqueductal gray (PAG) inhibits nociceptive inputs to sacral dorsal horn nociceptive neurons through alpha2-adrenergic receptors.

Modulation of sacral spinal dorsal horn neurons by the ventrolateral PAG was studied by extracellular recording combined with microiontophoretic applications of alpha-adrenergic agonists or antagonists. Bicuculline (BIC, 15 ng) microinjected into the ventrolateral PAG produced a consistent inhibition of the responses of nociceptive dorsal horn neurons. After PAG-BIC applications, the total number of spikes per heat stimulation period was significantly decreased to a mean of 37 +/- 19% (n = 8) of the pre-BIC control. Local iontophoresis of the selective alpha2-adrenoceptor antagonists idazoxan or yohimbine but not the selective alpha1 antagonist benoxathian significantly reversed PAG-BIC-evoked inhibition. At low ejection currents, clonidine, an alpha2-adrenoceptor agonist, markedly reduced noxious heat-evoked responses but had no consistent action on the responses to iontophoresed excitatory amino acids [EAA; N-methyl--aspartate (NMDA) or kainic acid]. At ejection currents higher than required to block descending inhibition, idazoxan potentiated responses to both heat and EAA iontophoresis. At higher ejection currents, EAA responses were inhibited by clonidine. This indicates that both presynaptic and postsynaptic alpha2 receptors are capable of inhibiting the recorded neurons. Activation of the alpha1 adrenoceptors by iontophoresis of methoxamine often led to a marked increase in the responses to kainic acid and, to a lesser extent, to NMDA iontophoresis or noxious heat. Together with previously reported work, the current experiments demonstrate that PAG neurons inhibit nociceptive dorsal horn neurons primarily through an indirect alpha2 adrenoceptor mechanism. In this same population of dorsal horn neurons, norepinephrine has a direct alpha1-mediated excitatory effect.

Endogenous opioid peptides acting at mu-opioid receptors in the dorsal horn contribute to midbrain modulation of spinal nociceptive neurons.

Activation of neurons in the midbrain periaqueductal gray (PAG) inhibits spinal dorsal horn neurons and produces behavioral antinociception in animals and analgesia in humans. Although dorsal horn regions modulated by PAG activation contain all three opioid receptor classes (mu, delta, and kappa), as well as enkephalinergic interneurons and terminal fields, descending opioid-mediated inhibition of dorsal horn neurons has not been demonstrated. We examined the contribution of dorsal horn mu-opioid receptors to the PAG-elicited descending modulation of nociceptive transmission. Single-unit extracellular recordings were made from rat sacral dorsal horn neurons activated by noxious heating of the tail. Microinjections of bicuculline (BIC) in the ventrolateral PAG led to a 60-80% decrease in the neuronal responses to heat. At the same time, the responses of the same neurons to iontophoretically applied NMDA or kainic acid were not consistently inhibited. The inhibition of heat-evoked responses by PAG BIC was reversed by iontophoretic application of the selective mu-opioid receptor antagonists, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP). A similar effect was produced by naloxone; however, naloxone had an excitatory influence on dorsal horn neurons in the absence of PAG-evoked descending inhibition. This is the first demonstration that endogenous opioids acting via spinal mu-opioid receptors contribute to brain stem control of nociceptive spinal dorsal horn neurons. The inhibition appears to result in part from presynaptic inhibition of afferents to dorsal horn neurons.

The involvement of metabotropic glutamate receptors in sensory transmission in dorsal horn of the rat spinal cord.

The role of metabotropic glutamate receptors in the processing of somatosensory information was studied in dorsal horn neurons of the rat spinal cord. Activation of metabotropic glutamate receptors by local iontophoresis of (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid resulted in an increased response of dorsal horn neurons to ionotropic glutamate receptor agonists (N-methyl-D-aspartate and kainic acid) applied by iontophoresis. Greater amounts of 1S,3R-1-amino-cyclopentane-1,3-dicarboxylic acid, ejected at high iontophoresis currents, directly excited dorsal horn neurons. Application of (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid also led to a significant increase in responses to innocuous (brush, pressure) but not in responses to noxious (pinch, squeeze) mechanical stimulation. The excitatory effects of (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid were selectively blocked by (S)-4-carboxy-3-hydroxyphenyl-glycine, an antinociceptive phenylglycine derivative which is a selective group 1 metabotropic glutamate receptor antagonist, confirming the involvement of these receptors. In wide dynamic range neurons, wind-up, the progressive potentiation of C-fibre-evoked responses during a train of stimuli, was increased by iontophoretic application of (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid or decreased by iontophoresis of (S)-4-carboxy-3-hydroxyphenyl-glycine without significant change in the C-fibre input. The results suggest an interaction between metabotropic and ionotropic glutamate receptors in spinal dorsal horn neurons. Metabotropic glutamate receptors proved to be involved in the frequency-dependent potentiation of C-fibre responses possibly via modulation of ionotropic glutamate receptors. The long-lasting effects of (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid on wind-up and on responses to peripheral mechanical stimuli strongly support the view that metabotropic glutamate receptors in these neurons may play a significant role in spinal synaptic plasticity, and therefore, may contribute to the central sensitization during mechanical hyperalgesia.

Spinal NK1 receptors contribute to the increased excitability of the nociceptive flexor reflex during persistent peripheral inflammation.

Hyperalgesia is a characteristic of inflammation and is mediated, in part, by an increase in the excitability of spinal neurons. Although substance P does not appear to mediate fast synaptic events that underlie nociception in the spinal cord, it may contribute to the hyperalgesia and increased excitability of spinal neurons during inflammation induced by complete Freund's adjuvant. We examined the role of endogenous substance P in changes in the excitability of spinal neurons during adjuvant-induced, peripheral inflammation by determining the effect of a selective NK1 receptor antagonist (RP67580) on the nociceptive flexor reflex in adult rats. Experiments were conducted 2 or 3 days after injection of adjuvant. Animals exhibited moderate thermal hyperalgesia at this time. The flexor reflex was evoked by electrical stimulation of the sural nerve and was recorded in the ipsilateral hamstring muscles. The flexor reflex ipsilateral to the inflamed hindpaw was enhanced approximately two-fold compared to the flexor reflex evoked in untreated animals as determined by the number of potentials and the duration of the reflex. The enhanced reflex in adjuvant-treated animals was most likely due to an increase in the excitability of spinal interneurons because short-latency activity in the hamstring muscles did not differ between untreated animals and adjuvant-treated animals following electrical stimulation of the L5 dorsal root or the nerve innervating the muscle with a stimulus that was 1.3-1.5 times the threshold for excitation of A-fibers. Intrathecal administration of RP67580 (2.3 and 6.8 nmol) attenuated the flexor reflex evoked in adjuvant-treated animals, but had no effect in untreated animals. Intravenous or intraplantar injection of RP67580 (6.8 nmol) did not affect the flexor reflex in adjuvant-treated animals indicating a spinal action of the drug following intrathecal administration. RP68651, the enantiomer of RP67580, was without effect at doses up to 6.8 nmol, indicating that the effects of comparable doses of RP67580 were due to an action of the drug at NK1 receptors. However, intrathecal administration of 23 nmol of both drugs attenuated the reflex in adjuvant-treated and control animals indicating that effects of RP67580 at this dose were not mediated entirely by its action at NK1 receptors. Overall, these data suggest that endogenous substance P has a role in the increased excitability of spinal interneurons observed during persistent inflammation and support the hypothesis that substance P released in the spinal cord contributes to the hyperalgesia that accompanies adjuvant-induced persistent, peripheral inflammation.

Role of substance P in the modulation of C-fiber-evoked responses of spinal dorsal horn neurons.

Substance P (SP) as well as excitatory amino acids (EAAs) appear to be released in response to stimulation of primary afferent C-fibers. Activity at N-methyl-D-aspartate (NMDA) receptors is essential for wind-up (the progressive potentiation of C-fiber-evoked responses of single neurons in response to an electrical stimulation), however, the role of SP in wind-up is unclear. To address this, the effects of iontophoretically applied CP-99,994 (a NK-1 receptor antagonist), SP and SP(1-7) (an N-terminal breakdown product of SP), were compared on responses of spinal dorsal horn wide dynamic range (WDR) neurons of the rat. Post-stimulus time histograms (PSTH) were summed over 12 responses to low frequency (0.5 Hz) electrical stimulation of the cutaneous receptive field. Changes in responses of dorsal horn neurons were evaluated by monitoring C-fiber input, wind-up, and the total number of spikes evoked by C-fiber activity in response to the 12 stimuli. The NK-1 receptor antagonist CP-99,994 significantly inhibited the total number of C-spikes and caused a significant reduction in wind-up without changing the C-fiber input, indicating the involvement of NK-1 receptors in wind-up. Application of SP led to an overall increase in the total number of C-fiber evoked responses of dorsal horn neurons and C-fiber input, however, wind-up, as defined, was significantly decreased following SP. In contrast, substance P(1-7) evoked a long-lasting increase in the total number of C-fiber-related spikes which was initially sustained by a transient increase in the input followed by a longer lasting increase in wind-up, an effect opposite that of CP-99,994. As NMDA activity has been previously shown to be inhibited and then potentiated by SP N-terminal activity over a similar time interval, the present data are consistent with the mediation of wind-up by NMDA and its modulation by SP N-terminal activity. Release of SP in response to noxious stimulation may, therefore, increase primary afferent C-fiber activity (input) whereas an accumulation of SP N-terminal metabolites appears to potentiate wind-up, perhaps via positive modulation of EAA activity.

Effects of nitric oxide availability on responses of spinal wide dynamic range neurons to excitatory amino acids.

The role of nitric oxide (NO) in responses of spinal dorsal horn neurons to excitatory amino acids and to cutaneous mechanical stimuli was examined. Extracellular recordings were made from wide dynamic range neurons excited with iontophoretically applied excitatory amino acid agonists, N-methyl-D-aspartate (NMDA) and (R,S)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) or kainic acid. Nitric oxide availability was decreased by iontrophoretic application of NO synthase inhibitors, N omega-nitro-L-arginine methyl ester (L-NAME) or L-N5-(1-iminoethyl)ornithine (L-NIO), or elevated by the NO donating compound, S-nitroso-N-penicillamine (SNAP). When cells were excited with successive application of NMDA and non-NMDA excitatory amino acid receptor agonists, application of NO synthase inhibitors led to a decrease in responses to NMDA in 60% of neurons. In more than a third of the cells tested, inhibition of NO synthase caused reciprocal changes in responses to glutamate receptor agonists: NMDA-evoked responses were significantly decreased whereas responses to the non-NMDA receptor agonists (AMPA or kainic acid) were increased. Application of the NO donating compound, S-nitroso-N-penicillamine, revealed an opposite tendency, increasing responses to NMDA in more than half of the neurons tested. In approximately 40% of the cells, reciprocal changes in responses to excitatory amino acid receptor agonists of NMDA versus non-NMDA types were observed after application of S-nitroso-N-penicillamine, such that the increase in NMDA responses was accompanied by decreases in the responses to kainic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

GYKI 52466 inhibits AMPA/kainate and peripheral mechanical sensory activity.

The selectivity of the 2,3-benzodiazepine compound, GYKI 52466, was tested on wide dynamic range (WDR) dorsal horn neurons of the rat spinal cord. Using extracellular recordings, neurons were characterized by stimulation with noxious and innocuous intensities of the receptive field. In most cells, responses to iontophoretically applied (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) or kainic acid (KA), but not N-methyl-D-aspartate (NMDA), were profoundly reduced by iontophoretic ejection of GYKI 52466. The inhibition usually lasted for 5-30 min following application of GYKI 52466. In a few neurons, responses to NMDA were also decreased by GYKI 52466. Responses to both noxious and innocuous mechanical stimulation were reduced in the presence of GYKI 52466. The results provide evidence for the selective inhibition by GYKI 52466 of AMPA/KA receptor-mediated functions and support the involvement of these receptors in spinal mechanical nociception.

A computer-controlled system for post-stimulus time histogram and wind-up studies.

A LabVIEW 2 virtual instrument (VI)-based system is described for stimulation of sensory fibers in peripheral nerves and analysis and storage of electrophysiological data recorded from the spinal cord. The system utilizes a commercially available multifunction data acquisition (input/output; I/O) board, graphical programming environment and custom built VIs. Electrical stimuli are triggered by the I/O board and delivered at C-fiber strength to the receptive field of a multireceptive dorsal horn neuron. The stimulus frequency can be chosen between 0.1 and 1 Hz. Extracellular action potentials are recorded with a compound recording/iontophoresis carbon-fiber microelectrode and sampled by the I/O board. Post-stimulus time histograms (PSTH) are computed to visualize the preferred times of neuronal firing evoked by A- and C-fiber inputs. Responses of the neuron to repetitive stimulation are analyzed by their latency relative to the stimulus and plotted against the stimulus number. Wind-up, the progressive potentiation of C fiber-evoked response per stimulus, is increased by NMDA and decreased by MK-801 when each is applied by microiontophoresis. The system is well suited to perform combined PSTH and wind-up analysis on-line as well as to examine previously recorded data off-line. Principles of operation and diagrams of greater importance are explained in detail. To illustrate system operation, sample recordings, PSTH and wind-up analyses from a dorsal horn neuron are presented. The software is available from the author.

Expression of the acetylcholinesterase gene during development of drosophila embryos.

The acetylcholinesterase (AChE) forms and the expression of the AChE mRNA in situ have been shown during the embryonic development of Drosophila melanogaster. The enzyme and its transcript were present well before the differentiation of the first neuroblasts. The non-CNS- specific AChE forms were responsible for the early AChE activity, and specific AChE form to the central nervous system (CNS) appeared when the CNS started to condense. Embryos deficient for the 5' end of the AChE gene expressed only the non-CNS-specific AChE forms,-- interestingly, the AChE transcript was present only in their CNS. The elimination of half of the CNS-specific AChE elevated the acetylcholine (ACh) level in the flies. These results imply that the non-CNS-specific AChE can also be non-neural, it is dispensable for the late embryonic development, and it does not substitute for the ACh hydrolysing capacity of the CNS- specific enzyme.

An iconographic program for computer-controlled whole-cell voltage clamp experiments.

A Macintosh-based system is described for performing instrument control, data acquisition and storage operations in single-electrode whole-cell voltage clamp experiments. The system consists of a commercially available voltage clamp amplifier, multifunction input/output (I/O) board, graphical programming language (LabVIEW 2) and custom built 'virtual instrument' (VI). The I/O board is capable of fast (up to 110 ksample/s) multichannel analog-to-digital (A/D) conversion with 12-bit resolution. It can control the gain settings of the clamp unit through digital I/O lines and generate the P/N leak subtraction protocol to eliminate the linear portion of capacitive currents using analog output voltages and gating pulses. Complete voltage clamp protocols can be implemented using the on-screen front panel controls of the VI. It enables the user to visualize the acquired data, to graph sets of current-voltage (I-V) relations or to fit single-exponential functions to one current trace. To evaluate the adequacy of whole-cell recording, the total membrane capacitance (Cm), the series resistance (Rs) and the time constant (tau c) of the decay of the capacitive current are calculated using the single-exponential function fit to the data. The system is particularly well suited to the study of large quantities of transmembrane I-V relationships. Source code for the crucial elements of the VI as well as sample recordings from a cultured spinal cord neuron, illustrating system operation, are presented.

Acetylcholine level in the brain and other organs of the bivalve Anodonta cygnea L. and its modification by heavy metals.

Acetylcholine was detected and measured in the ganglia (60-80 nmol/g), in the heart (10-15 nmol/g) and in the adductor muscles (4-5 nmol/g) of the bivalve Anodonta cygnea L. using gas chromatographic determination. Treatment of the animals with low concentration of heavy metals, which cause change in the behaviour, resulted in decrease of the brain acetylcholine level. Within 7 days treatment Cu2+ caused 80 per cent, Cd2+ and Pb2+ 30 per cent reduction with varying types of recovery after wash.

Seasonal variations in acetylcholine content and the levels of cholinergic enzymes in the alimentary tract and heart of Rana esculenta L.

The acetylcholine (ACh) content and the relative levels of choline-acetyltransferase (ChAT) and acetylcholinesterase (AChE) were measured in the stomach and intestinal musculature and in the heart muscles of frogs (Rana esculenta L.) during hibernation (January-stage 1), low activity (April-stage 2) and high activity period (July-stage 3). The ACh content doubled in the stomach from stage 1 to 2, then little change occurred. In the intestine, the ACh content changed little during the stages, while slightly decreased levels were found in the heart from stage 1 to 3. A significant increase in the level of ACh was found in the brain during the entire period. The level of ChAT increased in all tissue samples during the time. The AChE level was not significantly different during the stages in the stomach and intestine. In the heart, a 2.5 fold increase was measured in April compared with January, followed by a sharp decrease to half of the April level by July. In the brain, the activity of this enzyme increased continuously from January to July. The results show that these important cholinergic parameters change differentially in different organs in this poikilotherm animal during the year, presumably resulting in a fine control of the viscera, probably in cooperation with other autonomic transmitter systems.

Modulation of N-methyl-D-aspartate and (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) responses of spinal nociceptive neurons by a N-terminal fragment of substance P.

The effects of an N-terminal fragment of substance P, substance P-(1-7) [SP-(1-7)], on the responses of dorsal horn nociceptive neurons to N-methyl-D-aspartate (NMDA) and (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) were tested by combined single-unit extracellular recordings/microiontophoresis. While SP-(1-7) had no effects when applied by itself, it was a potent and long-lasting modulator of both NMDA- and AMPA-mediated excitation of spinal dorsal horn nociceptive neurons. NMDA responses were transiently decreased (by an average of 36% of control at minimum) by SP-(1-7) followed by a more sustained increase (by 76% at maximum). In contrast, AMP responses were only increased by SP-(1-7) (by 81% at maximum). It is hypothesized that the actions of SP-(1-7) on excitatory amino acid (EAA) responses of dorsal horn nociceptive neurons reflect a novel mechanism by which SP and EAAs interact to modulate pain transmission.

Enhancement of NMDA-evoked neuronal activity by glycine in the rat spinal cord in vivo.

The effects of iontophoretically administered N-methyl-D-aspartate (NMDA), glycine and strychnine on nociceptive dorsal horn neurons of the rat spinal cord were studied to test the hypothesis that their responses to NMDA are influenced in vivo by glycine acting at a strychnine-insensitive site. Experiments were carried out on 44 dorsal horn neurons responsive to microiontophoretic application of NMDA and peripheral stimulation. Glycine alone either enhanced or inhibited NMDA responses depending upon its dose (151% and 68% of control, respectively). Strychnine alone increased the NMDA-induced neural firing (129%), suggesting the presence of endogenous glycine. When glycine was co-ejected with strychnine, NMDA responses were further elevated (171%) revealing the activation of strychnine-insensitive binding sites. These data provide evidence that glycine can potentiate NMDA responses of nociceptive dorsal horn neurons in vivo and thus that glycine sites on NMDA receptors on these neurons are not saturated.

Comparative study of acetylcholine synthesis in organs of freshwater teleosts.

The activity of acetylcholine (ACh) synthesizing enzyme, choline acetyltransferase (ChAT), and the content of ACh were determined in the brain, heart, red trunk muscle and midgut of 12 freshwater teleost species belonging to eight families: carp (Cyprinus carpio), tench (Tinca tinca), silver carp (Hypophthalmichtys molitrix), bighead (Hypophthalmichtys nobilis), wels (Silurus glanis), cat fish (Ictalurus nebulosus), eel (Anguilla anguilla), rainbow trout (Oncorhynchus mykiss), pike (Esox lucius), pike-perch (Stizosteidon lucioperca), ruffe (Acerina cernua) and pumpkinseed (Lepomis gibbosus). The rate of ACh degradation in the same tissues was characterized by measuring the activity of acetylcholinesterase (AChE). Comparisons by statistical evaluation of cholinergic parameters in the corresponding organs were made between the species or families. The highest ACh concentration was found in the brain (between 9.6 and 16.0 nmol/g), and the gut (4.6 to 17.4 nmol/g), followed by the heart (1.4 to 3.9 nmol/g) and trunk muscle (1.2 to 3.8 nmol/g). Good correlations were found between the ACh content and the ChAT activity in all the organs studied. The correlation coefficients were 0.92 and 0.72 for the brain and heart, respectively. The activity of ChAT and the amount of ACh found in the brain and trunk muscle varied considerably from one family to another, but was of comparable degree within one family. It was concluded that differences in the cholinergic parameters may reflect different feeding and swimming behavioral patterns.

Stimulation intensity as critical determinant of presynaptic receptor effectiveness.

A number of reports in the literature suggest that there is an inverse correlation between the intensity of nerve activation and the effectiveness of presynaptic receptors that inhibit transmitter release. This common feature of many presynaptic inhibitory receptors may provide important insights into the mechanism by which transmitter release is controlled. Sue Duckles and Dénes Budai discuss the implications such a relationship between the pattern of nerve activation and effectiveness of presynaptic receptors has for understanding the physiological role of neuromodulators in general.

Low volume perfusion-superfusion system for measurement of transmitter release from blood vessels in vitro.

An in vitro technique has been developed for the simultaneous recording of vasoconstriction and transmitter release from small perfused blood vessels. To avoid excessive dilution of the released substances, 6-12-mg segments of rabbit ear arteries or rat tail arteries were mounted in 0.5-mL tissue chambers and perfused-superfused in a closed system. Drug administration and removal were performed by timer-controlled solenoid valves. Oscillation in the baseline generated by the roller pump was dampened by delivering Kreb's solution through a 4-mL buffer vial filled partly with air. Vasoconstriction was elicited by electrical stimulation and detected by pressure sensitive transducers. Transducer-generated electrical signals were amplified, digitalized, and recorded by a computerized system. Release of norepinephrine was measured after the tissue was preloaded with [3H]norepinephrine. This system allowed us to measure minimum vasoconstrictions of 1-2 mm Hg and [3H]norepinephrine efflux evoked by 4-8 electrical pulses. There was a good linear correlation (r = 0.98) between the contractile responses and tritiated norepinephrine release. Endogenous norepinephrine released by electrical stimulation into the perfusion media was determined by high-performance liquid chromatography (HPLC) in the low picogram range (80-100 pg). Our novel experimental setup permits computerized data acquisition and automated in vitro pharmacological experiments on isolated blood vessels.