Paweł Szulczyk

Functional properties of lumbar preganglionic neurones

Lumbar preganglionic neurones projecting through WRL2 and L3 to lumbar ganglia caudal to L4 were investigated for those functional properties which are typical for postganglionic vasoconstrictor neurones supplying muscle and skin and for post ganglionic sudomotor neurones. The properties tested were the cardiac rhythmicity of the activity and the reactions to systemic hypoxia, to noxious stimulation of skin and (in part of the experiments) to vibrational stimuli. Furthermore, resting activity and conduction velocities of the asons were measured. 426 neurones were investigated. 311 (73%) of them were silent and could -- as far as tested -- not be excited by the afferent stimuli used. The conduction velocities of the axons of these neurones ranged from 0.5 to about 16 m/sec. 115 neurones had resting activity of 0.1--4.6 impulses/sec. The conduction velocities of their axons ranged from 0.5 to about 12 m/sec. 80 preganglionic neurones with resting activity were classified on the basis of the reflexes in these neurones to afferent stimuli. Preganglionic neurones reacting like postganglionic vasoconstrictor neurones to muscle (excited by systemic hypoxia and/or by noxious stimulation of skin; with cardic rhythmicity) were classified as type 1 neurones (26 from 80 neurones tested). The resting activity of these neurones was 1.8 +/- 1.3 impulses/sec (mean +/- 1 S.D.). Their axons conducted with 3.9 +/- m/sec. Preganglionic neurones reacting like the majority of the postganglionic vasoconstrictor neurones to hairy and hairless skin (inhibited by systemic hypoxia and/or noxious cutaneous stimuli) were classified as type 2 neurones (48 from 80 neurones investigated). In 40% of these neurones the activity had cardiac rhythmicity. The resting activity was 0.9 +/- 0.6 impulses/sec. The distribution of the conduction velocities of the axons of these neurones was bimodal. They conducted on the average with 1.3 +/- 0.6 m/sec and 6.6 +/- 1.1 m/sec respectively. A few neurones were found (6 from 80 neurones) which were activated by vibrational stimuli (activation of Pacinian corpuscles by tapping on the hindfoot). Since this type of activation is typical for postganglionic sudomotor neurones they were classified as type 3 neurones. The activity of these neurones had no cardiac rhythmicity. Indirect measurements of the conduction velocities of pregnanglionic axons converging onto postganglionic neurones supplying skeletal muscle and hairy skin yielded values which were statistically not different from the conduction velocities of the axons of type 1 and type 2 neurones respectively. These measurements support the classification into type 1 and type 2 preganglionic neurones. The implications of this study are discussed.

D1 DOPAMINERGIC CONTROL OF G PROTEIN-DEPENDENT INWARD RECTIFIER K+ (GIRK)-LIKE CHANNEL CURRENT IN PYRAMIDAL NEURONS OF THE MEDIAL PREFRONTAL CORTEX

Pyramidal neurons of the medial prefrontal cortex (mPFC) exhibit dopamine-dependent prolonged depolarization, which may lead to persistent activity. Persistent activation of prefrontal cortex neurons has been proposed to underlie the working memory process. The purpose of our study was to test the hypothesis that activation of D(1) dopamine receptors leads to inhibition of G protein-dependent inward rectifier K(+) (GIRK) channels, thereby supporting the prolonged depolarization of mPFC pyramidal neurons. Experiments were performed on 3-week-old rats. GIRK-like channel currents recorded from pyramidal neurons showed the following properties at -75 mV: open probability (NPo), 2.5+/-0.3 x 10(-3); mean open time, 0.53+/-0.05 ms; and conductance, 29.9+/-1.6 pS (n=60). The channel currents were strongly inward-rectified. GIRK channel currents were reversibly inhibited by the D(1) agonists SKF 38393 (10 microM) and SKF 81297 (10 microM). This inhibition was abolished by prior application of a dopamine receptor antagonist and by application of the membrane-permeable protein kinase C inhibitors chelerythrine chloride (3 microM) and calphostin C (10 microM). It was also found that the application of D(1) dopamine receptor agonists or GIRK channel inhibitors evoked depolarization of mPFC pyramidal neurons in rats. Moreover, prior application of a GIRK channel blocker eliminated the depolarizing effect of D(1) agonists. We conclude that activation of D(1) dopamine receptors may lead to inhibition of GIRK channel currents that may, in turn, lead to the prolonged depolarization of mPFC pyramidal neurons in juvenile rats.

Opioid μ receptor activation inhibits sodium currents in prefrontal cortical neurons via a protein kinase A- and C-dependent mechanism

Opioid transmission in the medial prefrontal cortex is involved in mood regulation and is altered by drug dependency. However, the mechanism by which ionic channels in cortical neurons are controlled by mu opioid receptors has not been elucidated. In this study, the effect of mu opioid receptor activation on voltage-dependent Na(+) currents was assessed in medial prefrontal cortical neurons. In 66 out of 98 nonpyramidal neurons, the application of 1 microM of DAMGO ([D-Ala(2), N-Me-Phe(4), Gly(5)-OL]-enkephalin), a specific mu receptor agonist, caused a decrease in the Na(+) current amplitude to approximately 79% of that observed in controls (half blocking concentration = 0.094 microM). Moreover, DAMGO decreased the maximum current activation rate, prolonged its time-dependent inactivation, and shifted the half inactivation voltage from -63.4 mV to -71.5 mV. DAMGO prolonged the time constant of recovery from inactivation from 5.4 ms to 7.4 ms. The DAMGO-evoked inhibition of Na(+) current was attenuated when GDP-beta-S (0.4 mM, Guanosine 5-[beta-thio]diphosphate trilithium salt) was included in the intracellular solution. Inhibitors of kinase A and C greatly attenuated the DAMGO-induced inhibition, while adenylyl cyclase and kinase C activators mirrored the DAMGO inhibitory effect. Na(+) currents in pyramidal neurons were insensitive to DAMGO. We conclude that the activation of mu opioid receptors inhibits the voltage-dependent Na(+) currents expressed in nonpyramidal neurons of the medial prefrontal cortex, and that kinases A and C are involved in this inhibitory pathway.

Spinal segmental preganglionic outflow to cervical sympathetic trunk and postganglionic cardiac sympathetic nerves.

The aim of this study was to verify in which spinal cord segments the preganglionic neurones projecting to the cervical sympathetic trunk or converging onto the somata of the postganglionic cardiac sympathetic neurones are located in cats. The thoracic white rami T1 to T5 were electrically stimulated and the evoked responses were recorded in the cervical sympathetic trunks and postganglionic cardiac nerves. The responses were mostly evoked by electrical stimulation of group B preganglionic fibres. The maximum amplitude of evoked responses in the cervical sympathetic trunk was obtained when the T2 white ramus was stimulated and decreased gradually when followed by the stimulation of T1, T3, T4 and T5 white rami. In most cases the maximum amplitude of evoked responses in the left inferior cardiac nerve, right inferior cardiac nerve and left middle cardiac nerve was obtained when the T3 white ramus was stimulated. The size of the responses decreased when more cranial and caudal white rami were stimulated. It was found that the somata of the postganglionic neurones of the right and left inferior cardiac nerves were placed in the right and left stellate ganglion, respectively. Somata of the postganglionic neurones with axons in the left middle cardiac nerve were mainly located in the left middle cervical ganglion and some in the left stellate ganglion.

Spinal segmental sympathetic outflow to cervical sympathetic trunk, vertebral nerve, inferior cardiac nerve and sympathetic fibres in the thoracic vagus

The spinal segmental localization of preganglionic neurons which convey activity to the sympathetic nerves, i.e. vertebral nerve, right inferior cardiac nerve, sympathetic fibres in the thoracic vagus and cervical sympathetic trunk, was determined on the right side in chloralose anaesthetized cats. For that purpose the upper thoracic white rami were electrically stimulated with a single pulse, suprathreshold for B and C fibres, and the evoked responses were recorded in the sympathetic nerves. The relative preganglionic input from each segment of the spinal cord to the four sympathetic nerves was determined from the size of the evoked responses. It was found that each sympathetic nerve receives a maximum preganglionic input from one segment of the spinal cord (dominant segment) and that the preganglionic input gradually decreased from neighbouring segments. The spinal segmental preganglionic outflow to the cervical sympathetic trunk, thoracic vagus, right inferior cardiac nerve and vertebral nerve gradually shifted from the most rostral to the most caudal spinal cord segments. In some cases, a marked postganglionic component was found in the cervical sympathetic trunk. It was evoked by preganglionic input from the same spinal cord segments which transmitted activity to the vertebral nerve. These results indicate that there is a fixed relation between the spinal segmental localization of preganglionic neurons and the branch of the stellate ganglion receiving the input from these neurons.

Stellate neurones innervating the rat heart express N, L and P/Q calcium channels

The aim of the study was to investigate the kinetic properties and identify the subtypes of Ca2+ currents in the cardiac postganglionic sympathetic neurones of rats. Neurones were labelled with a fluorescent tracer--Fast-Blue, injected into the pericardial cavity. Voltage-dependent Ca2+ currents were recorded from dispersed stellate ganglion cells that showed Fast Blue labelling. Only high threshold voltage-dependent Ca2+ currents were found in the somata of cardiac sympathetic neurones. Their maximum amplitude, mean cell capacitance and current density were respectively: 0.67 nA, 19.3 pF and 36.4 pA/pF (n = 21). The maximum Ca2+ conductance was 51.3 nS (n = 14). Half activation voltage equalled +11.0 mV and the slope factor for conductance 11.1 (n = 14). As tested with a 10 s pre-pulse, the Ca2+ current began to inactivate at -80 mV. Half inactivation voltage and slope factor for steady-state inactivation were -36.6 mV and 14.1 (n = 9), respectively. Saturating concentration of L channel blocker (nifedipine), N channel blocker (omega-conotoxin-GVIA), P/Q channel blocker (omega-Agatoxin-IVA) and N/P/Q channel blocker (omega-conotoxin-MVIIC) reduced the total Ca2+ current by 26.8% (n = 7), 57.1% (n = 12), 25.9% (n = 6) and 69.4% (n = 6), respectively. These results show that the somata of cardiac postganglionic cardiac sympathetic neurones contain significant populations of N, L and P/Q high threshold Ca2+ channels.

Synthesis and anticonvulsant activity of novel 2,6-diketopiperazine derivatives. Part 1: perhydropyrrole[1,2-a]pyrazines.

A number of novel pyrrole[1,2-a]pyrazine derivatives were synthesized and evaluated in in vivo animal models of epilepsy. Among them, several compounds displayed promising seizure protection in the maximal electroshock seizure (MES), subcutaneous metrazol seizure (scMET), 6 Hz and pilocarpine-induced status prevention (PISP) tests, with ED(50) values comparable to the reference anticonvulsant drugs (AEDs). A critical influence of the stereochemistry and conformational preferences of the pyrrole[1,2-a]pyrazine core on in vivo pharmacological activity was observed. The mechanism of the anticonvulsant action of the agents synthesized is most probably not via inhibition of the voltage-dependent sodium (Na(+)) currents.

Expression and kinetic properties of Na(+) currents in rat cardiac dorsal root ganglion neurons.

The expression and properties of voltage-gated Na(+) currents in cardiac dorsal root ganglion (DRG) neurons were assessed in this study. Cardiac DRG neurons were labelled by injecting the Fast Blue fluorescent tracer into the pericardium. Recordings were performed from 138 cells. Voltage-dependent Na(+) currents were found in 115 neurons. There were 109 neurons in which both tetrodotoxin-sensitive (TTX-S, blocked by 1 microM of TTX) and tetrodotoxin-resistant (TTX-R, insensitive to 1 microM of TTX) Na(+) currents were present. Five cells expressed TTX-R current only and one cell only the TTX-S current. The kinetic properties of Na(+) currents and action potential waveform parameters were measured in neurons with cell membrane capacitance ranging from 15 to 75 pF. The densities of TTX-R (110.0 pA/pF) and TTX-S (126.1 pA/pF) currents were not significantly different. Current threshold was significantly higher for TTX-R (-34 mV) than for TTX-S (-40.4 mV) currents. V(1/2) of activation for TTX-S current (-19.6 mV) was significantly more negative than for TTX-R current (-9.2 mV), but k factors did not differ significantly. V(1/2) and the k constant for inactivation for TTX-S currents were -35.6 and -5.7 mV, respectively. These values were significantly lower than those recorded for TTX-R current for which V(1/2) and k were -62.3 and -7.7 mV, respectively. The action potential threshold was lower, the 10-90% rise time and potential width were shorter before than after the application of TTX. Based on this we drew the conclusion that action potential recorded before adding tetrodotoxin was mainly TTX-S current dependent, while the action potential recorded after the application of toxin was TTX-R current dependent. We also found 23 cells with mean membrane capacitance ranging from 12 to 35 pF (the smallest labelled DRG cells found in this study) that did not express the Na(+) current. The function of these cells is unclear. We conclude that the overwhelming majority of cardiac dorsal root ganglion neurons in which voltage-dependent Na(+) currents were present, exhibited both TTX-S and TTX-R Na(+) currents with remarkably similar expression and kinetic properties.

Properties of Na+ currents in putative submandibular and cardiac sympathetic postganglionic neurones

This study was performed to compare the kinetic properties of Na+ currents in putative salivary and cardiac postganglionic sympathetic neurones isolated from the superior cervical and stellate ganglia, respectively. Neurones were labelled with a fluorescent tracer-Fast Blue, injected into the submandibular gland (in the case of salivary neurones) and into the pericardial cavity or left ventricular wall (in the case of cardiac neurones). Voltage-dependent Na+ current was then isolated and recorded from labelled cells. The major findings of this study were: (1) Peak Na+ current was larger in salivary than in cardiac neurones (5.7 nA vs. 2.4 nA; for 30 mM Na+ in extra- and 15 mM in the intracellular solution). (2) The somata of salivary neurones were twice as large as those of cardiac neurones, as indicated by the values of their membrane capacitance (36 pF vs. 18 pF). (3) There was a greater Na+ current density (169 pA/pF vs. 128 pA/pF) in salivary than in cardiac neurones. (4) Recovery from inactivation was faster in salivary neurones with 90% recovery time being 93 ms for salivary and 144 ms in cardiac neurones. (5) Half-activation times were voltage-dependent and consistently longer for salivary than for cardiac neurones. (6) Remaining parameters, such as current threshold, maximum current voltage and kinetics of steady-state inactivation did not significantly differ in salivary compared to cardiac neurones.

Voltage-dependent k+ currents in rat cardiac dorsal root ganglion neurons

We have assessed the expression and kinetics of voltage-gated K(+) currents in cardiac dorsal root ganglion (DRG) neurons in rats. The neurons were labelled by prior injection of a fluorescent tracer into the pericardial sack. Ninety-nine neurons were labelled: 24% small (diameter<30 microm), 66% medium-sized (diameter 30 microm>.48 microm) and 10% large (>48 microm) neurons. Current recordings were performed in small and medium-sized neurons. The kinetic and pharmacological properties of K(+) currents recorded in these two groups of neurons were identical and the results obtained from these neurons were pooled. Three types of K(+) currents were identified:a) I(As), slowly activating and slowly time-dependently inactivating current, with V(1/2) of activation -18 mV and current density at +30 mV equal to 164 pA/pF, V(1/2) of inactivation at -84 mV. b) I(Af) current, fast activating and fast time-dependently inactivating current, with V(1/2) of activation at two mV and current density at +30 mV equal to 180 pA/pF, V(1/2) of inactivation at -26 mV. At resting membrane potential I(As) was inactivated, whilst I(Af), available for activation. The I(As) currents recovered faster from inactivation than I(Af) current. 4-Aminopiridyne (4-AP) (10 mM) and tetraethylammonium (TEA) (100 mM) produced 98% and 92% reductions of I(Af) current, respectively and 27% and 66% of I(As) current, respectively. c) The I(K) current that did not inactivate over time. Its V(1/2) of activation was -11 mV and its current density equaled 67 pA/pF. This current was inhibited by 95% (100 mM) TEA, whilst 4-AP (10 mM) produced its 23% reduction. All three K(+) current components (I(As), I(Af) and I(K)) were present in every small and medium-sized cardiac DRG neuron. We suggest that at hyperpolarized membrane potentials the fast reactivating I(As) current limits the action potential firing rate of cardiac DRG neurons. At depolarised membrane potentials the I(Af) K(+) current, the reactivation of which is very slow, does not oppose the firing rate of cardiac DRG neurons.