Leech Central Nervous System Research Articles

Evidence that glial cells modulate extracellular pH transients induced by neuronal activity in the leech central nervous system.

1. The role of the giant neuropile glial cells in the buffering of activity-related extracellular pH changes was studied in segmental ganglia of the leech Hirudo medicinalis L. using pH-sensitive microelectrodes and a slow, two-electrode voltage-clamp system. Neuronal activity was induced by electrical stimulation of a ganglionic side nerve (20 Hz, 1 min). 2. In CO2-HCO3(-)-buffered saline the glial cells were depolarized by 6.5 +/- 2.3 mV and alkalinized by 0.024 +/- 0.006 pH units (mean +/- SD) during the stimulation. The stimulation induced an acidification of 0.032 +/- 0.006 pH units in the extracellular spaces (ECS). 3. Voltage clamping the glial cells suppressed the stimulus-induced glial depolarization and turned the intraglial alkalinization into an acidification of 0.045 +/- 0.021 pH units (n = 6) that closely resembled the acidification observed in the presence of the anion transport blocker DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid, 0.5 mM), and in CO2-HCO(3-)-free saline. 4. Voltage clamping the glial cell resulted in the appearance of a distinct stimulus-induced extracellular alkalinization of 0.024 +/- 0.013 pH units at the onset of the stimulation, as also observed during DIDS application and in the absence of CO2-HCO3-. 5. The results suggest that glial uptake of bicarbonate is mediated by depolarization-induced activation of the electrogenic Na(+)-HCO3- cotransport, which suppresses the profound alkalinization of the ECS during neuronal activity. This is the first direct evidence the glial cells actively modulate extracellular pH changes in a voltage-dependent manner.

Neuronal responses to purinoceptor agonists in the leech central nervous system.

Extracellular nucleotides like ATP and its derivatives are possible chemical messengers in vertebrate nervous systems. In invertebrate nervous system, however, little is known about their role in neurotransmission. We have studied the response of identified neurones of the leech Hirudo medicinalis to the purinoceptor agonist ATP, ADP, AMP, and adenosine using conventional intracellular microelectrodes and whole-cell patch-clamp recording. Bath application of the agonists depolarized the different neurons, but not the neuropil glial cells. The most effective responses (up to 10 mV) were observed with ATP (100 microM) or ADP (100 microM) in the noxious and touch cells. In most neurons the nonhydrolyzable ATP derivative ATP-gamma-S (5 microM) induced larger depolarizations than 100 microM ATP, indicating that most of the potency of ATP is lost presumably due to its degradation by ectonucleotidases. In medial noxious cells, ATP (100 microM) induced an inward current of 1.7 +/- 1.1 nA at a holding potential of -60 mV. The ATP-induced current-voltage relationship showed an inward rectification and a reversal potential close to 0 mV. In a Na+-free extracellular solution, the ATP-induced inward current decreased and in a Na+- and Ca(2+)-free saline only a small residual current persisted. The possible P2 purinoceptor antagonist suramin did not antagonize the ATP-induced current, but itself evoked an inward current and a conductance increase. We conclude that ATP activates nonselective cation channels in medial noxious cells of the leech with the order of potency of purinoceptor agonists ATP > or = ADP > AMP. The results suggest that these cells express purinoceptors of the P2 type.

Membrane properties of microglial cells isolated from the leech central nervous system.

Membrane potentials and channel properties of microglial cells isolated from the leech central nervous system and maintained in culture on different substrates were investigated by using the patch-clamp technique. As expected, microglia on concanavalin A (con-A) were round and stationary, whereas those on extract of extracellular matrix (ECM) were spindle shaped and mobile. The mean membrane capacitance was 9 +/- 1 pF (s.e., n = 46), and the input resistance ranged from 0.135 G omega to 21 G omega with a mean of 4.2 +/- 1.6 G omega (n = 19). On-cell patches exhibited no single-channel activity. Voltage-dependent Na+, K+ and Ca2+ currents typical of neurons were absent. Currents evoked in response to voltage ramps from -100 mV to +100 mV or to steps of 4 s duration reversed in sign at or near 0 mV and exhibited single-channel activity of increasing amplitude for incrementally larger positive and negative voltage steps. No differences between the membrane properties of microglial cells on con-A and on ECM were evident. Currents were increased in fluid in which Na+ was substituted with K+, and were decreased when Na+ was substituted with N-methyl-D-glucamine. Varying external [Cl-] was without effect, as was addition to the fluid of 100 microM anthracene-9-carboxylate, a Cl- channel blocker. Together these characteristics indicated a cation channel. Bath application of 100 microM serotonin reversibly increased both inward and outward currents as well as single-channel activity. It is concluded that cultured microglial cells isolated from the adult leech have high membrane resistance and cation channel activity influenced by serotonin.

Interleukin-1 and interleukin-6 modify protein phosphorylation in the central nervous system of Hirudo medicinalis

Recent data suggest that IL-1-like molecules have been conserved during evolution. The signal transduction mechanism of IL-1 is not known, but kinase activation has been reported. With the aim of studying if human IL-1 has any effect on the leech nervous system, we have added this cytokine to segmental ganglia labeled with [ 32P]ortophosphoric acid; proteins have been separated by electrophoresis and phosphoproteins detected by autoradiography. In the present paper we show that human IL-1 and IL-6 are able to induce changes on protein phosphorylation in the leech central nervous system and that these changes are similar to those ones induced by the neurotransmitter serotonin.

Molecules that become redistributed during regeneration of the leech central nervous system.

A search has been made for molecules other than laminin that change their distribution during axonal regrowth after injury. Two monoclonal antibodies, generated against a protein extract of leech central nervous system (CNS), stain distinct regions of leech CNS and, following lesions, show changes in distribution with time. 1. Monoclonal antibody NP17 stained two bands of M(r) 80 x 10(3) and 60 x 10(3) on Western blots of protein extracted from whole CNS. On cryosections of leech CNS, staining was predominantly within the neuropile. 2. A second antibody, mAb CT16, stained a collagen-associated molecule of the extracellular matrix. It labelled the outer capsule, which surrounds the neuronal cell packets, and the inner capsule, which envelopes the neuropile. In the connectives, CT16 immunoreactivity was restricted to the connective capsule and was not associated with nerve fibres or glia. 3. When the connectives were crushed, immunocytochemical studies revealed changes in distribution of both mAb NP17 and mAb CT16. After 3 days, a time when fibres begin to sprout and form connections, an increase in NP17 immunoreactivity appeared at the site of the lesion. Staining levels remained elevated for several weeks. In contrast, CT16 immunoreactivity did not change for several days after damage. After 10 days, fibre-like staining appeared at the site of the crush; for several weeks it continued to spread throughout the connective. 4. These results show that regeneration of the leech CNS involves the redistribution of at least two molecules. Using monoclonal antibodies, these two molecules, which are situated in distinct regions of the CNS, have been visualized at different stages of the repair process. It has been shown that they alter their distribution at distinct times during regeneration.

Identification of a 70 kD protein with sequence homology to squid neurofilament protein in glial cells of the leech CNS.

A monoclonal antibody G39, generated against a protein extract of leech central nervous system, labels specific cell types in adult, embryonic, and regenerating preparations. The antibody stained glial cells, microglial cells, and connective tissue cells, but not neurons or muscle on cryosections. The staining pattern resembled that of an intracellular network. Affinity purification of the antigen revealed a 70 kD protein. Peptide sequencing showed significant homology of a stretch of 15 amino acids to squid neural filament protein. The same mAb G39 delineated glial cells as they formed during development of the CNS and showed that the giant neuropil glial cells appear before those in the packets. The antigen recognized by mAb G39 represents a nonneuronal intermediate filament of the leech Hirudo medicinalis found in various cell-types such as glia, microglia, and some cells of the connective tissue.

Single ion channel currents in neuropile glial cells of the leech central nervous system.

The patch-clamp technique was used to investigate the activity of single ion channels in neuropile glial (NG) cells in the central nervous system (CNS) of the medicinal leech, Hirudo medicinalis. We found evidence for two distinct Cl- channels that could be distinguished by their basic electrical properties and their responses to different inhibitors on single ion channel currents. In the inside-out configuration in symmetrical Cl- solutions, these channels showed current-voltage relationships with slight outward rectification, mean conductances of 70 and 80 pS, and reversal potentials near 0 mV. Significant permeability to Na+, K+, or SO4(2-) could not be detected. The open-state probability of the 70 pS Cl- channel increased with membrane depolarization, whereas the open-state probability of the 80 pS Cl- channel was voltage-independent. The application of the stilbene derivative DIDS (100 microM) to the cytoplasmic side of the glial cell membrane blocked both Cl- channels. The activity of the 70 pS channel was blocked irreversibly by DIDS, whereas the activity of the 80 pS channel reappeared after wash-out of DIDS. Both channels were blocked reversibly by 1 mM Zn2+. K+ channels could only be observed occasionally in the soma membrane of the NG cells. We have characterized a 60 pS K+ channel with a high selectivity for K+ over Na+. The low density of K+ channels in the soma membrane may indicate a non-uniform distribution of this channel type in NG cells.

Evidence for two isoforms of carbonic anhydrase II in the leech ( Hirudo medicinalis) central nervous system

1. 1. We dissected, homogenized and prepared ganglia and connectives from the central nervous system of medicinal leeches for SDS gel electrophoresis. The isolated proteins were transferred to nitrocellulose and incubated with affinity column-purified antibodies. 2. 2. The immunoblots showed a strong positive reaction of a bovine carbonic anhydrase standard at a molecular weight of 29 kDa, and a distinct double-bond at the same molecular weight in the analyzed material. 3. 3. We demonstrated with rat antibodies that carbonic anhydrase II is detectable in the leech central nervous system as the main isoenzyme. 4. 4. The biochemical knowledge of carbonic anhydrase reported here agrees well with the immunocytochemical locations, thus affirming the validity of specific staining.

Axonal sprouting and laminin appearance after destruction of glial sheaths.

Laminin, a large extracellular matrix molecule, is associated with axonal outgrowth during development and regeneration of the nervous system in a variety of animals. In the leech central nervous system, laminin immunoreactivity appears after axon injury in advance of the regenerating axons. Although studies of vertebrate nervous system in culture have implicated glial and Schwann cells as possible sources, the cells that deposit laminin at sites crucial for regeneration in the living animal are not known. We have made a direct test to determine whether, in the central nervous system of the leech, cells other than ensheathing glial cells can produce laminin. Ensheathing glial cells of adult leeches were ablated selectively by intracellular injection of a protease. As a result, leech laminin accumulated within 10 days in regions of the central nervous system where it is not normally found, and undamaged, intact axons began to sprout extensively. In normal leeches laminin immunoreactivity is situated only in the basement membrane that surrounds the central nervous system, whereas after ablation of ensheathing glia it appeared in spaces through which neurons grew. Within days of ablation of the glial cell, small mobile phagocytes, or microglia, accumulated in the spaces formerly occupied by the glial cell. Microglia were concentrated at precisely the sites of new laminin appearance and axon sprouting. These results suggest that in the animal, as in culture, leech laminin promotes sprouting and that microglia may be responsible for its appearance.

PH recovery from intracellular alkalinization in Retzius neurones of the leech central nervous system.

1. Neutral-carrier pH-sensitive microelectrodes were used to investigate intracellular pH (pHi) recovery from alkalinization in leech Retzius neurones in Hepes- and in CO2-HCO3(-)-buffered solution. The Retzius neurones were alkaline loaded by the addition and subsequent removal of 16 mM acetate, by changing from 5% CO2-27 mM HCO3- to 2% CO2-11 mM HCO3- or by changing from CO2-HCO3(-)- to Hepes-buffered solution. 2. In Hepes-buffered solution (pH 7.4) the mean pHi was 7.29 +/- 0.11 and the mean membrane potential -44.7 +/- 5.9 mV (mean +/- S.D.; n = 83). 3. The rate of pHi recovery from alkalinization increased with decreasing pH of the bathing medium (pHb). pHi changed about 0.30 pH units for a pHb unit change. 4. A decrease of extracellular buffer concentration (Hepes concentration lowered from 20 to 5 mM) caused an acidification of extracellular and intracellular pH and an acceleration of pHi recovery from alkalinization. 5. A depolarization of the Retzius cell membrane-induced by increasing the K+ concentration of the bathing medium from 4 to 20 mM (delta Em = 16.5 +/- 5.5 mV) or from 4 to 40 mM (delta Em = 24.8 +/- 3.5 mV)--evoked a decrease of pHi and an acceleration of pHi recovery from alkalinization. 6. The H+ current blocker Zn2+ (0.5 mM) inhibited pHi recovery from alkalinization at resting membrane potential as well as during depolarization. The inhibition was more pronounced during depolarization. 7. In Cl(-)-free, CO2-HCO3(-)-buffered solution pHi recovery from an alkaline load by changing from 5% CO2-27 mM HCO3- to 2% CO2-11 mM HCO3- was slowed by 48-71%. The rate of pHi recovery from an alkaline load induced by changing from CO2-HCO3- to Hepes buffer was reduced by 33-56% in Cl(-)-free solution. The removal of external Cl- did not affect pHi recovery in Hepes-buffered solution. 8. The pHi recovery from alkalinization was DIDS-insensitive in CO2-HCO3(-)- as in Hepes-buffered solutions and was not slowed in the absence of external Na+. 9. It is concluded that in Retzius neurones pHi recovery from alkalinization is mediated by a passive voltage-dependent H+ influx along the electrochemical proton gradient. In the presence of CO2-HCO3- buffer a DIDS-insensitive Cl(-)-HCO3- exchanger additionally regulates pHi after an intracellular alkaline load. It cannot be excluded that intracellular processes (e.g. H+ release from organelles, metabolic H+ production) are also involved in pHi recovery from alkalinization.

Glutamate-like immunoreactivity in the leech central nervous system.

Using a monoclonal antibody for glutamate the distribution was determined of glutamate-like immunoreactive neurons in the leech central nervous system (CNS). Glutamate-like immunoreactive neurons (GINs) were found to be localized to the anterior portion of the leech CNS: in the first segmental ganglion and in the subesophageal ganglion. Exactly five pairs of GINs consistently reacted with the glutamate antibody. Two medial pairs of GINs were located in the subesophageal ganglion and shared several morphological characteristics with two medial pairs of GINs in the first segmental ganglion. An additional lateral pair of GINs was also located in segmental ganglion 1. A pair of glutamate-like immunoreactive neurons, which are potential homologs of the lateral pair of GINs in segmental ganglion 1, were occasionally observed in more posterior segmental ganglia along with a selective group of neuronal processes. Thus only a small, localized population of neurons in the leech CNS appears to use glutamate as their neurotransmitter.

The role of matrix molecules in regeneration of leech CNS.

Extracellular matrix (ECM) molecules extracted from the leech central nervous system (CNS) provide substrates that induce extensive growth of processes of identified leech nerve cells in culture. Two ECM molecules, laminin and tenascin, have been identified. The laminin-like molecule has been purified and shown to be a cross-shaped molecule similar to vertebrate laminin with subunits of 340, 220, 180, and 160 kD. Purified laminin as a substrate induces rapid outgrowth of Retzius (R) and Anterior Pagoda (AP) cells in culture. The tenascin molecule has been partially purified. In electronmicrographs, leech tenascin, like vertebrate tenascin, has six arms of equal size joined in a central globule. Highly enriched fractions of leech tenascin induce rapid and extensive outgrowth of Retzius and AP cells in culture. Substrate molecules not only induce outgrowth of processes but also affect the growth patterns of individual nerve cells. Neurites are straight with few branches in laminin, but curved with profuse branches on tenascin. During regeneration of the CNS in the animal, laminin appears at new sites associated with growth cones. The appearance of laminin correlates with the accumulation of microglial cells. Thus, ECM molecules with growth-promoting activity for leech nerve cells in vitro appear to be involved in inducing regeneration and allowing the neurites to reconnect with former targets.

The regulation of pH in the central nervous system.

The pHi regulation from intracellular acidosis in the central nervous system appears to be mediated by mechanisms driven by the large inwardly directed Na+ gradient. The involvement of these mechanisms in pHi regulation of neurones and glial cells has been investigated in the leech central nervous system using ion-selective microelectrodes. For recovery from acidification, there appear to be three separate mechanisms: Na+/H+ exchange, Na(+)-dependent Cl-/HCO3- exchange, and Na+-HCO3- cotransport. All three mechanisms have a profound effect on the maintenance of pHi homeostasis in glial cells; whereas in leech neurones, as in other neuronal cells studied previously, the predominant mechanisms are Na+/H+ and Na(+)-dependent Cl-/HCO3- exchange. In addition to acid extrusion mechanisms we also found evidence for Na(+)-independent Cl-/HCO3- exchange. At alkaline pHi this exchanger may mediate some of the pHi recovery from intracellular alkalinization.

Synaptogenesis and calcium current distribution in cultured leech neurons

The distribution of calcium (Ca$^{+}$) currents was studied by loose-patch technique in Retzius cells isolated from the leech central nervous system. Ca$^{+}$ tail currents were recorded in patches held at the equilibrium potential for potassium (E$_{\text{K}}$). Measurements were made from single cells and paired cells that had formed chemical or electrical synapses in culture. In single Retzius cells, Ca$^{+}$ currents in the stump of the initial process were three times larger than in the middle part of the soma; intermediate amplitudes were found in the polar region of the soma. After the formation of chemical synapses, Ca$^{+}$ currents at the stumps of postsynaptic cells and in the polar region of the somas of presynaptic cells were reduced. The formation of electrical synapses did not affect Ca$^{+}$ current distribution.

Evidence for glial control of extracellular pH in the leech central nervous system.

Double-barrelled pH-sensitive microelectrodes were used to measure the intracellular pH (pHi) of neuropil glial cells and the pH of extracellular spaces (pH(e)) within isolated, intact ganglia of the leech Hirudo medicinalis. By application of a weak acid (propionate, 40 mM) or a weak base (ammonium, 20 mM) the total buffer capacity was estimated by changes in glial pHi and in pH(e). The buffering power of glial cells and in the extracellular spaces was increased by up to threefold in the presence of CO2/bicarbonate. The anion exchange inhibitor 4,4-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 0.3-0.5 mM) reversed this increase in buffering power both in the glial cells and in the extracellular spaces. Inhibitors of the carbonic anhydrases reduced the CO2/bicarbonate-dependent increase in extracellular buffering power. The results suggest that extracellular H+ buffering dependent upon the availability of bicarbonate is linked to DIDS-sensitive bicarbonate transport across the glial membrane.

Segmental differentiation in the leech central nervous system: Proposed segmental homologs of the heart accessory neurons

As part of an on-going study of segmental differentiation in the central nervous system (CNS) of the leech Hirudo medicinalis, a search was made for putative segmental homologs of the heart accessory (HA) neurons, which exist exclusively as a bilateral pair in the ganglia of the fifth and sixth body segments. As it is not yet feasible to obtain adequate cell lineage information in H. medicinalis, potential homologs of the HA neurons were determined using morphological, immunohistochemical, and electrophysiological criteria. Among cells in other body ganglia with somata in the same locations as HA neurons, a pair was found having extensive morphological and physiological similarities to HA neurons. These we have called HA-like (HAL) neurons. Adult HA and HAL neurons have closely related patterns of primary branching, in terms of shape, intraganglionic pathways taken, and extraganglionic projections. The number, location, and relative thickness of branches are also similar among these cells. In embryos 10 to 11 days old, HA and HAL neurons have virtually identical branching patterns, with primary and secondary branches of nearly uniform caliber. Differences in branch thickness develop gradually; by embryonic day 20, they resemble those found in adult neurons. Two features found to differ between HA and HAL neurons were the cell body diameter (larger for the HA cells) and the expression of antigens recognized by the monoclonal antibody Laz1-1 (absent at a detectable level in the HA neurons). At a physiological level, the HA and HAL neurons showed action potentials of similar size and shape, as well as inhibitory synaptic inputs from a common source, the heart interneurons (HN). The observations presented here suggest that there is a common developmental origin for the HA and HAL neurons, and hence that their fates are positionally determined by as yet unknown factors.

Electrogenic sodium-dependent bicarbonate secretion by glial cells of the leech central nervous system.

The ability to move acid/base equivalents across the membrane of identified glial cells was investigated in isolated segmental ganglia of the leech Hirudo medicinalis. The intracellular pH (pHi) of the glial cells was measured with double-barreled, neutral-ligand, ion-sensitive microelectrodes during step changes of the external pH (pHo 7.4-7.0). The rate of intracellular acidification after the decrease in extracellular pH (pHo) was taken as a measure of the rate of acid/base transport across the glial membrane. Taking into account the total intracellular buffering power, the maximum rate of acid/base flux was 0.4 mM/min in CO2/HCO3-free saline, and 3.92 mM/min in the presence of 5% CO2/10 mM HCO-3, suggesting that the acid/base flux was dependent upon HCO3-. The rate of acid influx/base efflux increased both with the external HCO3- concentration and with increasing pHi (and hence HCO3-i). This suggested that the decrease in pHi was due to HCO3- efflux. The rapid decrease of pHi was accompanied by a HCO3--dependent depolarization of the glial membrane from -74 +/- 5 mV (n = 20) to -54 +/- 7 mV (n = 13). Both this depolarization and the rate of intracellular acidification were greatly reduced by the anion exchange inhibitor 4,4-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 0.3-0.5 mM), but were not affected by the removal of external Cl-. Reduction of the external Na+ concentration to one-tenth normal affected the rate of intracellular acidification only in the presence of CO2/HCO3-: the rate increased within the first 3-5 min after lowering external Na+; after longer exposures in low external Na+ the rate decreased, presumably due to depletion of intracellular Na+. Amiloride (1 mM), which inhibits the Na+-H+ exchange in these cells, had no effect on the rate of intracellular acidification. The intracellular Na activity (aNai) of the glial cells was measured to be 5.2 +/- 1.0 mM (n = 8) in CO2/HCO3-free saline; aNai increased to 7.3 +/- 2.2 mM (n = 8) after the addition of 5% CO2/24 mM HCO3-. Upon a change in pHo to 7.0 in the presence of CO2/HCO3-, aNai decreased by an average of 2 +/- 1.1 mM (n = 5); in CO2/HCO3--free saline external acidification produced a transient increase in aNai. It is concluded that, in the presence of CO2/HCO3-, the rate of intracellular acidification in glial cells is dominated by an outwardly directed, electrogenic Na+-HCO3-cotransport. Neurons, which do not possess this cotransporter, acidify at much lower rates under similar conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Extracellular matrix molecules in development and regeneration of the leech CNS

As neurons grow to their targets their processes elongate, branch and form specialized endings into which are inserted appropriate ion channels. Our aim has been to analyse the role of the extracellular matrix molecules laminin and tenascin in inducing growth and in determining the form and physiological properties of growing neurites. A preparation in which development and regeneration can be followed at the cellular and molecular level in the animal and in tissue culture is the central nervous system (CNS) of the leech. In leech extracellular matrix (ECM) both laminin and tenascin are present; the molecules are structurally similar but not identical to their vertebrate counterparts. Tenascin extracted from leech ECM shows a typical hexabrachial structure whereas laminin shows a typical cruciform structure in rotary shadowed preparations. Leech laminin purified by means of a monoclonal antibody is a molecule of about 1000 kDa, with a polypeptide composition of 340, 200, 180 and 160 kDa. Substrates that contain tenascin or laminin produce rapid and reliable outgrowth of neurites by identified cells. A remarkable finding is that the outgrowth pattern produced by an individual neuron depends in part on its identity, in part on the substrate upon which it is placed. For example, a Retzius cell grows in a quite different configuration and far more rapidly on laminin substrate than does another type of neuron containing the same transmitter (serotonin); and the pattern of outgrowth of the Retzius cell is different on laminin and on the plant lectin Con A (concanavalin A). Thus Con A induces the growth of processes that are shorter, thicker, more curved and contain fewer calcium channels than those grown on laminin. To determine whether laminin can also influence neurite outgrowth in the animal, immunocytological techniques have been used to follow its distribution in the extracellular matrix of normal, developing and regenerating leech CNS. In adult leeches neuronal processes in the CNS are not in contact with laminin which is confined to the surrounding extracellular matrix. In embryos however, laminin staining appears between ganglionic primordia along the pathways that neurons will follow. Similarly, after injury to the adult CNS, laminin accumulates at the very sites at which sprouting and regeneration begin. How the laminin becomes redistributed to appear in the region of injury has not yet been established. Together these findings suggest a key role for laminin and for other extracellular matrix molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ouabain on intracellular ion activities of sensory neurons of the leech central nervous system

1. The intracellular K+, Na+, and Ca2+ of mechanosensory neurons in the central nervous system of the leech Hirudo medicinalis was measured using double-barreled ion-sensitive microelectrodes. 2. After inhibition of the Na(+)-K+ pump with 5 x 10(-4) M ouabain, the intracellular K+ activity (aKi) decreased, while the intracellular Na+ activity (aNai) increased. The input resistance decreased in the presence of ouabain. The intracellular Ca2+ increased more than one order of magnitude after ouabain addition. All changes in intracellular ion activities and membrane resistance were fully reversible. 3. When extracellular Na+ concentration ([Na+]o) was removed [replaced by tris(hydroxymethyl)aminomethane (Tris)], aNai decreased. In the absence of [Na+]o, aKi and aNai remained unchanged after inhibition of the Na(+)-K+ pump by reducing the extracellular K+ concentration ([K+]o) to 0.2 mM. The membrane resistance increased under these conditions. 4. The intracellular Ca2+ decreased or remained constant after removal of [Na+]o. Addition of ouabain in the absence of [Na+]o did not change intracellular Ca2+, which only increased after readdition of [Na+]o. 5. The relative K+ permeability (PK) measured as membrane potential change during a brief increase of the [K+]o from 4 to 10 mM, increased manyfold after addition of ouabain but only little if [Na+]o had been removed before adding ouabain. 6. The results suggest that the intracellular Na+ increase after inhibition of the Na(+)-K+ pump affects the intracellular Ca2+ level by stimulating a Nai(+)-Ca2+ exchange mechanism. The subsequent intracellular Ca2+ activity (aCai) rise may result in an increase of the membrane permeability to K+ ions.

Serotonin in the leech central nervous system: anatomical correlates and behavioral effects

1. Serotonin is sequestered by a limited population of identified neurons in the 32 ganglia of the leech nervous system. A major fraction of the serotonin in each ganglion is contained in the paired Retzius cells, colossal effector neurons whose size varies longitudinally. The 5 other classes of identified serotonin-containing neurons, one effector cell and 4 interneurons, are approximately twice as numerous in anterior as in posterior ganglia. 2. We dissected 6 longitudinal samples from the ventral nerve cords of hungry Hirudo medicinalis, and measured their serotonin content using high pressure liquid chromatography with electrochemical detection. A consistent neurochemical pattern emerged in which segmental ganglia 2-4 had the highest quantity of serotonin: 18.51 pmol per ganglion. The anterior cerebral ganglion contained 14.78 pmol, and the content of the 4 posterior samples, segmental ganglia 7-10, 12-15, 17-20 and the caudal ganglion, decreased continuously from 16.35, 15.08, 10.75 to 2.51 pmol per ganglion, respectively. Morphometric analyses indicated that this pattern of ganglionic serotonin correlated primarily with longitudinal variations in the number of serotonin neurons per ganglion and secondarily with volume of the Retzius cells. Retzius cell volume correlated highly with the mass of their innervated body segments both of which are largest in mid-body domains. 3. Serotonin expresses leech feeding, and its ganglionic levels are a potentially useful index of behavioral state. We measured serotonin in the ganglionic samples from hungry and satiated leeches. The samples from recently fed animals contained 28% less serotonin than those from hungry ones. The amounts of serotonin in the cerebral and all the segmental samples from satiated leeches were significantly lower than equivalent samples of hungry animals. A similar pattern of depletion was seen in leeches which fed for a prolonged period (90 to 120 min) rather than the normal period of 30 min. 4. The effects of ingestion on serotonin-containing neurons was examined with the glyoxylic acid-induced histochemical fluorescence. The levels of fluorescence in all serotonin neurons in fed leeches were consistently lower than those in equivalent neurons in hungry animals, corroborating the ganglionic decrease in serotonin in satiated leeches. 5. To examine effects of body wall distension on serotonin levels, hungry leeches were fed to satiation, and half of them were relaxed by removing their distending blood meals. After 6 weeks, ganglionic serotonin in leeches with relaxed bodies was 21% higher than in those with distended bodies. 6. Ingestive behavior depletes serotonin from leech neurons and body wall distension appears to interfere with its synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)