Synaptic Blockade Research Articles

Chapter 3 Spinal neuronal thermosensitivity in vivo and in vitro in relation to hypothalamic neuronal thermosensitivity

In the spinal cord, temperature signals are generated which serve as specific inputs in the central nervous control of body temperature. Because of the spatially distinct organization of afferent and efferent neuronal systems at the spinal level, the afferent pathway for temperature signal transmission could be identified in vivo in the ascending, anterior and lateral tracts with a relationship of about 75:25% between warm and cold sensitive neuraxons. Analysis of spinal neuronal thermosensitivity in vitro on spinal cord tissue slices has been concerned, so far, with the superficial laminae of the dorsal horn as the site of origin of ascending nerve fibers conveying mostly temperature and pain signals, and with lamina X as a site of origin of afferent as well as efferent neurons. A relationship of about 95:5% between warm and cold sensitive neurons was found at the segmental level, indicating that warm sensitivity is the prevailing, primary property of spinal neurons, whereas cold sensitivity seems to be mainly generated by synaptic interaction as a secondary modality. Dynamic responses to temperature changes were frequently displayed in vitro at the spinal segmental level in lamina I + II but not in lamina X, even by neurons whose static activity was little influenced by local temperature. Dynamic thermosensitivity was found less frequently in ascending tract neuraxons and was not observed in hypothalamic neurons receiving temperature signal inputs from the spinal cord, and thus, does not seem to be relevant for the thermosensory function of spinal cord neurons, unlike peripheral warm and cold receptors. A majority of spinal warm sensitive neurons displayed both static and dynamic warm sensitivity as an inherent property after synaptic blockade. In the further analysis of spinal cord thermosensitivity, the in vitro approach permits application of the same electrophysiological and neuropharmacological methods as were established for the analysis of hypothalamic thermosensitivity. In addition, the topography of the spinal cord will provide additional structural and possibly histochemical information to characterize the functions of neurons independently of their thermal properties.

Nefazodone and depression.

Nefazodone is a fourth-generation antidepressant with novel serotonergic actions. It is a potent antagonist of synaptic 5-HT(2) receptors and a moderate blocker of synaptic 5-HT reuptake. Its mild synaptic blockade of norepinephrine is of little clinical importance. Besides its effectiveness in depression, nefazodone is effective in depression-related anxiety. Of all the antidepressants, nefazodone is least likely to induce sexual dysfunction. This drug increases sleep efficiency and reduces the number of nighttime awakenings. Nefazodone, compared with many other antidepressants, is unlikely to induce a withdrawal syndrome if abruptly stopped.

Effects of vasopressin and involvement of receptor subtypes in the rat central amygdaloid nucleus in vitro

Effects of arginine-vasopressin (AVP) on neurons in the central amygdaloid nucleus (ACe) were investigated with rat brain slice preparations using extracellular recording methods. Of 160 ACe neurons tested, 70 cells (44%) were excited and 9 cells (6%) were inhibited by bath application of AVP at 3×10 −7 M. The excitatory effects of AVP were dose-dependent and the threshold concentration was approximately 10 −10 to 10 −9 M. The excitatory effects of AVP persisted under blockade of synaptic transmission by perfusing with Ca 2+-free and high-Mg 2+ medium, whereas the inhibitory effects were abolished by synaptic blockade. AVP-induced effects were mimicked by a V 1-receptor agonist and completely blocked by a selective V 1-antagonist. V 2-agonist produced no effects on ACe neurons and V 2-antagonist had no effect on AVP-induced excitation. These results showed that the excitatory effect of AVP on ACe neurons was produced by a direct action through the V 1-receptors, whereas the inhibitory response of ACe neurons to AVP seemed to be produced by an indirect action. The results of this study suggest that AVP is involved in the amygdala-relevant functions as a neurotransmitter or a neuromodulator.

Characterization of dopamine-induced depolarization of prefrontal cortical neurons.

Dopamine (DA) has been reported to depolarize neurons in the prefrontal cortex (PFC). To further characterize this effect of DA, we made whole cell recordings from PFC pyramidal cells in rat brain slices. As reported previously, DA depolarized most PFC cells tested. This effect of DA was concentration-dependent and persisted in the presence of synaptic blockade, indicating a direct effect of DA on the recorded cell. During DA-induced depolarization, PFC neurons consistently showed an increase in excitability, suggesting that the depolarization is not directly related to DA-induced inhibition of PFC neurons previously observed in vivo. Surprisingly, the effect of DA was not mimicked or blocked by several commonly used DA agonists and DA antagonists. The alpha and beta antagonists phentolamine and alprenolol and the atypical antipsychotic drug clozapine also showed no significant effect on DA-induced depolarization. These results suggest that DA-induced depolarization may be mediated by a nonspecific mechanism. However, it remains possible that there exists a new type of DA receptors in the PFC not sensitive to classical DA agonists and antagonists, particularly given the fact that DA applied in the same manner depolarized only PFC neurons but not those in the striatum or the substantia nigra.

Peptide YY and the Y2 agonist PYY-(13-36) inhibit neurons of the dorsal motor nucleus of the vagus.

Peptide YY (PYY) is released by endocrine cells in the ileum in response to the presence of fatty acids in the intestinal lumen. Circulating PYY suppresses vagally mediated digestive functions as a consequence of direct action on neurons in the dorsal medulla. Recent evidence from our laboratory suggests that this PYY-mediated inhibition of digestion occurs because of peptide action at the Y2-type receptor in the dorsal medullary region encompassing vago-vagal reflex circuitry. The present study describes the effects of PYY and the specific Y2 agonist peptide PYY-(13-36) on neurons of the dorsal motor nucleus of the vagus (DMN) in both the 1) intact in vivo and 2) in vitro brain stem slice preparation. Our results show that 50% of DMN neurons recorded under in vivo or in vitro conditions, including synaptic blockade, are inhibited by the application of PYY or PYY-(13-36). Approximately 45% are not affected, and only approximately 5% are activated. These results suggest that one of the principal means by which PYY suppresses digestive functions is by the direct inhibition of cholinergic vagal efferent neurons of the DMN via action at a Y2 receptor.

Cell–cell coupling occurs in dorsal medullary neurons after minimizing anatomical-coupling artifacts

Dye (Lucifer Yellow) and tracer (Biocytin) coupling, referred to collectively as anatomical coupling, were identified in 20% of the solitary complex neurons tested in medullary tissue slices (120–350 μm) prepared from rat, postnatal day 1–18, using a modified amphotericin B-perforated patch recording technique. Ten per cent of the neurons sampled in nuclei outside the solitary complex were anatomically coupled. Fifty-eight per cent of anatomically coupled neurons exhibited electrotonic postsynaptic potential-like activity, which had peak-to-peak amplitudes of ≤7 mV, with the same polarity as action potentials; increased and decreased in frequency during depolarizing and hyperpolarizing current injection; was maintained during high Mg 2+–low Ca 2+ chemical synaptic blockade; and was measured only in anatomically coupled neurons. The high correlation between anatomical coupling and electrotonic postsynaptic potential-like activity suggests that Lucifer Yellow, Biocytin and ionic current used the same pathways of intercellular communication, which were presumed to be gap junctions. Anatomical coupling was attributed solely to the junctional transfer of Lucifer Yellow and Biocytin since potential sources of non-junctional staining were minimized. Specifically, combining 0.26 mM amphotericin B and 3–5% Lucifer Yellow produced a hydrophobic, viscous solution that did not leak from the pressurized pipette tip (≤3 μm outer diameter) submerged in artificial cerebral spinal fluid. Moreover, unintentional contact of the pipette tip with adjacent neurons that resulted in accidental staining, another source of non-junctional staining, was averted by continuously visualizing the tip prior to tight seal formation with infrared video microscopy, used here for the first time with Hoffman modulation contrast optics. During perforated patch recording, which typically lasted for 1–3 h, Lucifer Yellow was confined to the pipette, indicating that the amphotericin B patch was intact. However, once the patch was intentionally ruptured at the end of recording, the viscous, lipophilic solution entered the neuron resulting in double labeling. Placing a mixture of amphotericin B, Biocytin and Lucifer Yellow directly into the pipette tip did not compromise tight seal formation with an exposed, cleaned soma, and resulted in immediate (<1 min) steady-state perforation at 22–25°C. Hence, this adaptation of conventional perforated patch recording was termed “rapid perforated patch recording”. The possible functional implication of cell–cell coupling in the dorsal medulla oblongata in central CO 2/H + chemoreception for the cardiorespiratory control systems is discussed in the second paper of this set [Huang et al. (1997) Neuroscience, this volume].

Nicotinic receptor-mediated responses in relay cells and interneurons in the rat lateral geniculate nucleus

We used the in vitro whole-cell recording technique to study the nicotinic responses of relay cells and interneurons in the adult rat dorsal lateral geniculate nucleus, the thalamic nucleus that conveys visual signals from the retina to the cortex. These geniculate relay cells and interneurons were identified by their physiological and morphological properties. We found that, in the presence of a muscarinic antagonist, atropine, acetylcholine induced a depolarization in relay cells. A similar depolarization was induced by application of nicotine. These depolarizations were completely blocked by a nicotinic antagonist, hexamethonium, but were little affected by bath solution that contained tetrodotoxin and/or low calcium concentration to block synaptic transmission. This suggests that the depolarization is mediated directly by nicotinic receptors in relay cells. Application of nicotine also induced a depolarization in geniculate interneurons. The interneurons continued to exhibit a response to nicotine in the presence of synaptic blockade, although the time-course of the response was altered. The nicotinic responses in relay cells and interneurons shared many similar properties. Both exhibited desensitization, although this characteristic was much more pronounced in the interneurons. In both cell types, the nicotinic response activated a relatively linear conductance with a slight inward rectification. The reversal potential for the conductance was about −33 mV, which is consistent with a permeability to sodium and potassium ions. The reversal potential shifted negatively by 5–6 mV when the bath solution contained low calcium, which further suggests a permeability to calcium ions. Our results indicate that nicotinic receptors are present in both geniculate relay cells and interneurons. The nicotinic depolarization in relay cells may serve to enhance transmission of visual signals through the lateral geniculate nucleus as well as to contribute to a voltage-dependent shift in the response mode of geniculate relay cells from burst to tonic (single-spike) firing. The nicotinic depolarization in interneurons may provide an explanation for reports that activation of the cholinergic system can enhance inhibitory tuning in the lateral geniculate nucleus.

Effects of bioamines and peptides on neurones in the ventral nucleus of trapezoid body and rostral periolivary regions of the rat superior olivary complex: an in vitro investigation

Intracellular microelectrode recordings were made from single neurones of the ventral nucleus of trapezoid body and rostral periolivary regions in the rat auditory brainstem, using in vitro slice techniques. Bath application was used to examine the effects of putative neurotransmitters and neuromodulators on cell responses to constant depolarizing current pulsse. Noradrenaline exerted excitatory effects (increased firing rate) that were probably mediated by α-receptors, whereas inhibitory effects (decreased firing rate) were probably mediated by β-receptors. Serotonin also produced either excitatory or inhibitory effects in different cells. Of the neuroactive peptides, substance P and enkephalin were especially potent. Substance P was found to be exclusively excitatory and enkephalin was exclusively inhibitory. Choleycystokinin exerted either inhibitory or excitatory effects in a small percentage of cells. Somatostatin had only very weak or non-existent effects. These effects were able to be elicited under conditions of synaptic blockade, indicating they they were mediated by direct action on the cells in question. Most effects on firing rate were accompanied by either depolarization or hyperpolarization of the resting membrane potential although in many cases this change in membrane potential was small. Changes in cell access resistance were also relatively difficult to detect, but in the case of both noradrenaline and substance P, clear increases in cell access resistance were recorded in a number of cells. These could be obtained in the presence of tetrodotoxin, again indicating a direct action of these substances rather than an indirect action mediated via synaptic connections. Although the exact mechanisms of action remain to be investigated in each case, it is clear that neurones in this region of the auditory brainstem are potentially subject to a wide variety of modulatory influences that could be important in auditory processing.

Effect of pancreatic polypeptide on rat dorsal vagal complex neurons.

1. Pancreatic polypeptide (PP) microinjected into the dorsal vagal complex (DVC) elevates gastric activity through a vagal mechanism. Thus, it was hypothesized that PP alters the activity of nuclei comprising the DVC, i.e. the nucleus tractus solitarii (NTS) and the dorsal motor nucleus (DMN). 2. In vivo and in vitro approaches were used. For in vivo studies, micropipettes were used for recording and injecting vehicle or PP. Neurons were identified as NTS or DMN using orthodromic and antidromic activation, respectively, following vagal stimulation. Gastric-related DVC neurons were located using antral inflation. For in vitro studies, DMN neurons were recorded from medullary slices. 3. Of the twenty-eight NTS and DMN neurons identified, fifteen were activated, six inhibited and seven unaffected after PP microinjection. Forty-two gastric-related neurons were located in the DVC, of which twenty-five were stimulated by PP and seventeen exhibited no change. No gastric-related cells were inhibited. 4. For in vitro studies, 66% of DMN neurons were activated by PP (n = 27/47) while the remaining 33% were inhibited (n = 14/47). Similar results were obtained in normal or synaptic blockade media. 5. These results support the hypothesis that PP alters DVC neuronal activity, which may thereby lead to the previously observed alterations in gastric activity.

The effects of temperature and synaptic blockade in vitro on neurons of the horizontal limb of the diagonal band of Broca in the rat basal forebrain

We studied the thermosensitivity of neurons in the rat horizontal limb of the diagonal band of Broca (HDB) in vitro under normal conditions and under conditions of a low calcium/high magnesium synaptic blockade (SB). Of 52 HDB neurons tested, 34 neurons (65%) were warm-sensitive (WS), three neurons (6%) were cold-sensitive (CS), 11 neurons (21%) were temperature-insensitive (TI) and four additional neurons (8%) were both warm- and cold-sensitive (WS/CS). Of 34 neurons tested for thermosensitivity under SB, 11 were WS, 4 were CS and 19 were TI. Nearly half (48%) of the WS neurons maintained warm sensitivity under SB, 43% became TI and 9% became CS. Baseline firing rates of neurons significantly decreased during SB and then increased during recovery from SB. In addition, a distinct anatomical distribution of thermosensitive neurons was found in the HDB. The most ventral aspect of the HDB (interaural +0.9–1.3 mm) had proportionally fewer temperature sensitive neurons (65% vs. 88%) than areas more dorsal (interaural +1.3–1.7 mm), and only one of seven ventral HDB neurons (14%) remained thermosensitive during SB. In the dorsal HDB, 65% of the neurons maintained thermosensitivity during SB. These results demonstrate that the HDB contains inherently thermosensitive neurons, and that a difference in thermal characteristics exists between the ventral and dorsal HDB neurons.

Is the input to a GABAergic or cholinergic synapse the sole asymmetry in rabbit's retinal directional selectivity?

We examined contrast, direction of motion, and concentration dependencies of the effects of GABAergic and cholinergic antagonists, and anticholinesterases on responses to movement of On-Off directionally selective (DS) ganglion cells of the rabbit's retina. The drugs tested were curare and hexamethonium bromide (cholinergic antagonists), physostigmine (anticholinesterase), and picrotoxin (GABAergic antagonist). They all reduced the cells' directional selectivity, while maintaining their preferred-null axis. However, cholinergic antagonists did not block directional selectivity completely even at saturating concentrations. The failure to eliminate directional selectivity was probably not due to an incomplete blockade of cholinergic receptors. In a extension of a Masland and Ames (1976) experiment, saturating concentrations of antagonists blocked the effects of exogenous acetylcholine or nicotine applied during synaptic blockade. Consequently, a noncholinergic pathway may be sufficient to account for at least some directional selectivity. This putative pathway interacts with the cholinergic pathway before spike generation, since physostigmine eliminated directional selectivity at contrasts lower than those saturating responses. This elimination apparently resulted from cholinergic-induced saturation, since reduction of contrast restored directional selectivity. Under picrotoxin, directional selectivity was lost in 33% of the cells regardless of contrast. However, 47% maintained their preferred direction despite saturating concentrations of picrotoxin, and 20% reversed the preferred and null directions. Therefore, models based solely on a GABAergic implementation of Barlow and Levick's asymmetric-inhibition model or solely on a cholinergic implementation of asymmetric-excitation models are not complete models of directional selectivity in the rabbit. We propose an alternate model for this retinal property.

Development of locomotor activity induced by NMDA receptor activation in the lumbar spinal cord of the rat fetus studied in vitro

The development of neuronal circuits generating locomotor activity was characterized in an isolated lumbar spinal cord preparation from of fetal and neonatal rats. Locomotor activity induced by bath application of the NMDA receptor agonists, NMA and NMDA, was monitored from both sides of the corresponding lumbar ventral roots. Activation of NMDA receptors first evoked rhythmic motor activity at E15.5. NMA-induced rhythmic motor activity was not observed under synaptic blockade by TTX or cadmium ions, suggesting that this activity was evoked by synaptic drive from the interneuronal circuits in the spinal cord. At E15.5–E16.5, the rhythmic motor activity on both sides was synchronized. Phase relationship of the rhythmic motor activity between both sides was variable at E17.5–E19.5. The rhythmic motor activity was alternating on both sides at E20.5. Mid-sagittal splitting of the spinal cord did not affect the rhythm generation at all stages examined, suggesting the existence of independent rhythm-generating circuits on each side. The rhythmic motor activity in the presence of strychnine was synchronized on both sides at all stages examined. These results indicate that the changes in rhythm pattern are mediated by development of glycinergic inhibitory pathways, while the basic rhythm can be generated without the glycinergic inhibitory pathways.

Modulation of ganglion cell activity in the pineal gland of the rainbow trout: effects of cholinergic, catecholaminergic, and GABAergic receptor agonists.

Second order neurons within intact isolated pineal glands of the rainbow trout were explored by extracellular recordings to investigate modulatory effects of putative intrapineal neurotransmitters. Acetylcholine, dopamine, and norepinephrine were found to increase ganglion cell activity in a majority of cells tested. The excitatory effects of acetylcholine, dopamine, and norepinephrine were mimicked by muscarinic, dopamine D2, and beta-adrenergic receptor agonists and significantly increased with the applied light intensity, resulting in an attenuation of the ganglion cell response to light. GABA decreased discharge activity in most cells tested. This effect, which could be mimicked with the GABAA receptor agonist piperidine, was independent from the adaptive status. Acetylcholine and GABA were still active if applied during synaptic blockade with low Ca++ high Mg(++)-perfusion medium, whereas dopamine and norepinephrine exhibited no effects if applied during synaptic blockade, suggesting a differential cellular distribution of neurotransmitter receptors in the trout pineal gland. These data demonstrate that ganglion cell activity in the trout pineal gland is under the influence of several neurotransmitters, including acetylcholine, dopamine, norepinephrine, and GABA, which is in contrast to the originally proposed simple bineuronal transduction pathway from photoreceptors onto ganglion cells. Since the above-mentioned neurotransmitters are believed to be released from pineal interneurons, we may conclude that ganglion cell activity in the teleost pineal gland is, similarly to the retina, the product of photoreceptor signals and a modulatory active interneuronal system.

Presynaptic or postsynaptic location of receptors for angiotensin II and substance P in the medial solitary tract nucleus.

1. Microinjection of angiotensin (Ang) II or substance P (SP) into the medial nucleus tractus solitarii (nTS) produces similar decreases in arterial pressure and heart rate. We previously reported that some medial nTS neurons responsive to SP were also excited by Ang II, and that Ang II increased the release of SP from medulla slices. Both electrophysiological and anatomic data suggest that the cardiovascular effects of these peptides may be mediated by a common neuronal pathway consisting of SP-containing vagal afferent fibers with presynaptic Ang II receptors that innervate medial nTS neurons with SP receptors. To evaluate the validity of this model, we established the presynaptic or postsynaptic location of the receptors for Ang II and SP that mediate excitation of medial nTS neurons by determining the capacity of each peptide to activate the cell before and after blocking synaptic transmission in rat dorsal medulla slices. 2. Extracellular recordings were obtained from 55 medial nTS neurons responsive to Ang II or SP in 400-microns horizontal slices of the dorsal medulla. Neuronal excitation by Ang II and SP was tested before, during, and after reversal of synaptic blockade with low-Ca2+ (0.2 mM), high Mg2+ (5 mM) artificial cerebrospinal fluid (aCSF). Elimination of synaptically evoked short latency responses of the neuron to current pulses applied to afferent fibers in the solitary tract (TS) documented blockade of synaptic transmission by low-Ca2+ aCSF. In most cases, the basal firing rate of the cell increased slowly during perfusion with low-Ca2+ aCSF and stabilized after approximately 30 min at a higher level of spontaneous activity. Responses to the peptides and TS stimulation were also documented after synaptic blockade had been reversed by adding aCSF containing 2-mM Ca2+. 3. Of the 55 medial nTS neurons, 41 were responsive to Ang II; whereas, 50 of the 55 cells were responsive to SP. The neurons were divided into three subgroups on the basis of their responsiveness to Ang II and SP. Although most neurons were responsive to both Ang II and SP (n = 36), five other cells were excited only by Ang II, and 14 neurons were activated only by SP. Of the 55 neurons, 26 were also responsive to L-glutamate: 14 of 17 cells responsive to both Ang II and SP, all 5 neurons excited by Ang II but not by SP, and 7 of 10 neurons responsive only to SP were also excited by L-glutamate. The latency of the action potentials evoked by TS stimulation was much shorter in those neurons responsive only to Ang II (3.6 ms) than in cells excited by both Ang II and SP (6.8 ms) or responsive only to SP (7.4 ms). 4. In 21 of the 36 medial nTS neurons responsive to both Ang II and SP, Ang II continued to excite the cell when synaptic responses to TS stimulation were prevented by low-Ca2+ aCSF, but had no effect on the firing rate of the other 15 neurons during synaptic blockade. Excitation induced by Ang II was also prevented in two of the five medial nTS neurons responsive only to Ang II when synaptic transmission in the slice was blocked. Low-Ca2+ aCSF failed to prevent excitation by SP or L-glutamate in all medial nTS cells responsive to these agonists (n = 50 and n = 26, respectively). In contrast to these observations in medial nTS neurons, Ang II-induced excitation was not altered during synaptic blockade in any of the six dmnX cells studied. No responses to SP or L-glutamate were blocked in dmnX neurons, as also seen in the medial nTS. 5. When all medial nTS neurons responsive to Ang II were examined, the latencies of the response to TS stimulation were significantly shorter in those neurons with presynaptic Ang II receptors than in the group of cells with postsynaptic receptors. In addition, neurons with presynaptic Ang II receptors were distributed differently within the medial nTS than cells with postsynaptic Ang II receptors.(ABSTRACT TRUNCATED)

Analysis of contributions of acetylcholine and tachykinins to neuro-neuronal transmission in motility reflexes in the guinea-pig ileum.

1. The roles of acetylcholine (ACh) and tachykinins in neuro-neuronal transmission during ascending excitatory and descending inhibitory reflexes were studied by recording intracellular reflex responses of the circular muscle to physiological stimuli. Experiments were carried out in opened segments of guinea pig ileum in an organ bath that was partitioned so that three regions could be independently exposed to drugs. 2. Ascending excitatory reflexes evoked by either distension from the serosal side or compression of the mucosa were depressed by 55% and 85%, respectively, in the presence of hexamethonium (200 microM) and by 30% and 45%, respectively, by a desensitizing concentration of the selective NK3 receptor agonist, senktide (1 microM), in the chamber in which reflexes were initiated. Together, hexamethonium and senktide abolished responses to compression. A residual response to distension persisted. This was abolished by hyoscine (1 microM). 3. Hexamethonium (200 microM) abolished ascending reflexes when applied to the region between the stimulus and the recording sites, or to the recording chamber. 4. Descending reflex responses were reduced by 35% by synaptic blockade in the stimulus chamber with physiological saline containing 0.1 mM Ca2+ plus 10 mM Mg2+. Senktide (1 microM) in the stimulus chamber reduced distension reflexes to the same extent as synaptic blockade, whereas hexamethonium (200 microM) and hyoscine (1 microM) depressed responses by less than 20%. Responses to compression were reduced by 40% by senktide alone, while senktide and hexamethonium together reduced responses by 60%, an effect similar to synaptic blockade. Under these conditions, hyoscine in the stimulus chamber restored reflexes evoked by distension, but did not alter those evoked by mucosal compression. 5. Total synaptic blockade in the intermediate chamber, between stimulus and recording sites, reduced descending reflex responses by more than 90%. In contrast, hexamethonium (200 microM) had no effect and hyoscine (1 microM) reduced only the responses to distension (by 30%). Senktide (1 microM) depressed responses to both stimuli by approximately 80%. 6. Application of hexamethonium (200 microM) to the recording chamber depressed descending reflex responses to distension applied in the near stimulation chamber by 15%, but had no effect on responses to compression in the near chamber or to either stimulus applied in the far chamber. 7. Descending reflexes evoked by near chamber stimuli were unaffected by hyoscine (1 microM) or senktide (1 microM) applied to the recording chamber; hyoscine enhanced reflexes evoked by compression in the far chamber by 50%. 8. For the ascending excitatory reflex pathway, it is concluded that transmission from sensory neurones is mediated by ACh acting via both nicotinic and muscarinic receptors, and by tachykinins acting at NK3 receptors. Transmission from ascending interneurones appears to be predominantly via nicotinic receptors. The descending inhibitory pathways are more complex, and while transmission from sensory neurones involves nicotinic, muscarinic and NK3 receptor-dependent components, transmission from descending interneurones to inhibitory motor neurones is neither cholinergic nor due to tachykinins acting via NK3 receptors.

γ-Aminobutyric acid enhances the light response of ganglion cells in the trout pineal organ

Electrical recordings from achromatic second order neurons of intact superfused pineal organs of the rainbow trout were used to investigate the role of γ-aminobutyric acid (GABA) in the transmission of photoreceptor signals onto centrally projecting ganglion cells. Bath-applied GABA decreased the spike discharge rate of 98% of achromatic ganglion cells in a dose-dependent manner. GABA was also active if applied during synaptic blockade, demonstrating the presence of GABAergic receptors at the ganglion cell level. Responsiveness of ganglion cells to light was reversibly enhanced by GABA. The light response curve of ganglion cells, which was obtained by plotting spike rate versus light intensity, was significantly shifted to lower frequencies by GABA, indicating that GABA is an important inhibitory modulator of ganglion cell activity in the trout pineal organ.

In vitro recordings from area postrema neurons demonstrate responsiveness to adrenomedullin.

Adrenomedullin (ADM) is a recently discovered 52-amino acid peptide that exerts potent vasodilatory effects in the periphery and influences the control of body fluid balance when injected centrally. In this study extracellular single-unit recordings were obtained from 94 AP neurons in rat brain slices. Bath application of ADM (10(-7) M) excited 47% (32 of 68) of cells tested, and these effects were found to be dose dependent from 10(-7) to 10(-9) M. Excitation was maintained during synaptic blockade in a low-Ca2+ artificial cerebrospinal fluid solution, demonstrating direct actions of ADM on these neurons. The remaining cells were either unaffected (n = 25) or inhibited (n = 11) by ADM. ADM (10(-7) M) also influenced the spontaneous activity of 9 (7 inhibited, 2 excited) of 16 neurons located in the nucleus tractus solitarii (NTS). However, these effects could be eliminated during synaptic blockade, suggesting indirect actions of the peptide on NTS neurons. These data demonstrate that a specific population of CNS neurons within the AP are directly influenced by ADM and suggest that ADM may exert its effects on the central control of fluid balance through direct actions at this circumventricular organ.

Binding proteins on synaptic membranes for certain phospholipases A2 with presynaptic toxicity.

Many steps in the process of neurotransmitter release are vulnerable to various neurotoxins. Some of these presynaptic toxins exhibit phospholipase A2 (PLA2) activity. These neurotoxic PLA2s (or PLA2 neurotoxins) are members of a group of extracellular (or secreted) PLA2 proteins found in most if not all animals. Besides phospholipid metabolism, these PLA2s exhibit a variety of biological effects, including host defense, neurotoxicity (presynaptic and/or postsynaptic), myotoxicity, and alteration of coagulation, which may or may not be related to hydrolysis of phospholipids. Despite large differences in biological actions, the PLA2 chains of these proteins show high degrees of homology in the primary, secondary and possibly tertiary structures. A small number of these proteins, mostly isolated from the venoms of a number of snakes, act primarily at the presynaptic level to cause synaptic blockade by inhibiting the release of neurotransmitters, though most of them also produce postsynaptic toxicity and other effects. These presynaptic PLA2 toxins may be classified into three classes. The toxins differ in their subunit structures, but in every case, at least one subunit is an active PLA2 with M.W. of 12,000 to 16,000. Each toxin in the first class is a single-chained protein. In the second class, a toxin may comprise 2 to 4 homologous subunits associated noncovalently.

Generation of interspike intervals of rat carotid body chemoreceptors.

Hypoxia is well established to stimulate carotid body chemoreceptors leading to an increase in sinus nerve activity, but the mechanism by which the nerve activity increases is unresolved. It is generally assumed that spike initiation is solely dependent on presynaptic transmitter secretion, but, synaptic blockade with low calcium solutions causes little change in baseline nerve activity (although it does eliminate the hypoxia response) (Donnelly & Kholwadwala, 1992). This suggests that the nerve endings can initiate action potentials independent of glomus cell secretion. However, it is difficult to study this directly because of the small size of the nerve terminals and uncertainty regarding the site of action potential generation. The present study was undertaken to better understand the process of spike initiation by examination of the interspike interval pattern from rat chemoreceptors. The results show that spike generation during normoxia and during hypoxia stimulation is a Poisson-type random process, and this generator characteristic is highly sensitive to the Na+ channel agonist, veratridine.

Vasopressin actions on area postrema neurons in vitro.

The area postrema (AP) is a circumventricular organ located on the dorsal surface of the medulla. Substantial evidence suggests that the AP is an important site involved in cardiovascular regulation. Arginine vasopressin (AVP) is thought to act at the AP to increase the sensitivity of the baroreceptor reflex. We have therefore examined the effects of AVP on AP neurons with the use of extracellular single unit recordings in vitro. Coronal medullary brain slices (thickness = 400 microns) were obtained from male Sprague-Dawley rats and maintained in oxygenated artificial cerebrospinal fluid (aCSF). The slices were perfused with AVP (10(-8) to 10(-6) M), and the effect on single AP neurons was recorded. A total of 79 AP neurons was tested of which 50 (63.3%) were excited by AVP and 5 (6.3%) were inhibited, whereas the remaining 24 (30.3%) cells were unaffected. The excitatory effects of AVP were dose dependent: firing rate increased 92.6 +/- 25.8% at 10(-8) M, 289.4 +/- 53.9% at 10(-7) M, and 456.8 +/- 113.1% at 10(-6) M, respectively. We also examined whether these effects of AVP resulted from direct actions of this peptide on AP cells by testing if responses were retained during blockade of synaptic transmission (achieved by perfusion with a low Ca(2+)-high Mg2+ aCSF) in 11 cells excited by AVP. Nine of these cells were excited by AVP during such synaptic blockade. Finally, we demonstrated that the excitatory responses of five AP cells to AVP were all totally abolished by perfusion of slices with aCSF containing the V1 antagonist ([1-beta-mercapto-beta,beta-cyclopentamethylene propionic acid,2-(O-methyl)tyrosine]-Arg8-vasopressin; Peninsula Laboratories, 10(-6) M).(ABSTRACT TRUNCATED AT 250 WORDS)