Long duration ventral root potentials in the neonatal rat spinal cord in vitro; the effects of ionotropic and metabotropic excitatory amino acid receptor antagonists.

Long duration, primary afferent evoked ventral root potentials (VRP's) have been recorded in vitro from hemisected spinal cords prepared from 8-12-day-old rat pups. Single shock stimulation of a dorsal root at stimulus strengths sufficient to recruit C/group IV afferent fibres evoked a long duration (11.9 +/- 1.2 s) ipsilateral VRP in all preparations. This long duration VRP consisted of two components, (i) a slow wave, time to peak 137.0 +/- 5.1 ms, the amplitude of which was reduced to 8.7% of mean control value in the presence of the N-methyl-D-aspartate (NMDA) antagonist D-AP5 (40 microM), (ii) a prolonged wave with a time to peak of 2.0 +/- 0.2 s which was partially resistant to D-AP5 (40 microM). Both the slow and the prolonged waves were unaffected following superfusion with the metabotropic excitatory amino acid (EAA) receptor antagonist L-AP3 (100-200 microM). Low frequency (1-10 Hz) repetitive stimulation (20 s duration) of high threshold dorsal root afferents evoked a temporal summation of synaptic activity which generated a progressively depolarizing VRP. This cumulative VRP was graded with frequency of stimulation (0.89 +/- 0.13 to 1.25 +/- 0.19 mV). The cumulative VRP was followed by a post-stimulus depolarization which outlasted the period of repetitive stimulation by tens of seconds (47.6 +/- 8.4 to 91.2 +/- 19.9 s). In the presence of AP5 the amplitude of the cumulative VRP was depressed to 54.5 +/- 11.5% of control values when low frequency (1.0 Hz) stimulation was used. The proportion of the cumulative VRP resistant to D-AP5 increased as the frequency of stimulation was increased to 10 Hz. The decay time of the post-stimulus depolarization was unaffected by AP5. Neither the amplitude nor the post-stimulus depolarization of the cumulative VRP was affected by 200 microM L-AP3. It is suggested that both an AP5 sensitive and AP5 insensitive potential contribute to the long duration VRP evoked in the neonatal rat spinal cord following single shock high threshold afferent stimulation. Moreover, the AP5 insensitive prolonged depolarization is manifest following sustained low frequency stimuli and higher frequency inputs.     

The contribution of increases in extracellular potassium to primary afferent depolarization in the bullfrog spinal cord

To assess the extent to which depolarization by accumulated K+ contributes to the generation of primary afferent depolarization (PAD), the isolated bullfrog spinal cord was superfused with K+-rich Ringer solutions and the resultant dorsal root depolarizations were recorded extracellularly. Action potential blockade (with tetrodotoxin) did not reduce the K+-induced depolarization of primary afferents, indicating that the depolarization was generated locally in the region around the afferents. In this respect superfusion with K+-rich solutions adequately models the localized K+ accumulation which occurs physiologically during afferent activity. K+-induced depolarizations were decreased in the presence of 20 mM Mg2+; this effect was due to a direct decrease in the membrane response to K+ and not to blockade of K+-induced transmitter release onto primary afferents. The depolarization caused by a K+ concentration comparable to a maximum estimate of the K+ accumulating around afferent terminals following a single afferent volley was found to account for no more than about one-third of the DRP height. However, higher K+ levels, comparable to those resulting from high frequency afferent stimulation, caused large depolarizations of primary afferents, sometimes greater than the DRP amplitude. Therefore, K+-induced depolarization may contribute more significantly to PAD evoked by high frequency afferent activity.     

Synaptic activation of cardiac vagal neurons by capsaicin sensitive and insensitive sensory neurons

Little is known about the central circuitry involved in the sensory activation of cardioinhibitory vagal neurons (CVNs). To study the polysynaptic activation of CVNs from sensory neurons the postsynaptic currents in CVNs in the dorsal motor nucleus of the vagus (DMNX) were evoked by stimulation of the vagus nerve. In addition, the role of afferent A-fiber and C-fiber activation of CVNs was examined. CVNs were identified by a retrograde fluorescent tracer and were studied in an in vitro slice preparation using patch-clamp electrophysiology. Stimulation of the vagus nerve evoked excitatory postsynaptic currents in CVNs that were reversibly blocked by the NMDA antagonist D-2-amino-5-phosphonovalerate (AP5) and the non-NMDA antagonist 6-cyano-7-nitroquionoxaline-2,3-dione (CNQX). Vagal stimulation also evoked inhibitory postsynaptic currents (IPSCs) that were reversibly blocked by the GABA(A) antagonist gabazine. Capsaicin, which inactivates C-fibers, was used to examine the role of afferent A-fibers and C-fibers in the synaptic activation of CVNs. Capsaicin significantly (P<0.05) reduced the amplitude of evoked glutamatergic and GABAergic postsynaptic currents by 59% and 76%, respectively. The latency of the GABAergic response increased significantly (P<0.05) in the presence of capsaicin from 36+/-1 to 41+/-1 ms while the latency of the glutamatergic response (44+/-3 ms) was unaffected. There are three conclusions from this study. Stimulation of vagal afferents evokes both GABAergic and glutamatergic responses in CVNs, C-type afferent fibers are critical to the afferent stimulation of CVNs, and the A-fiber GABAergic pathway to CVNs may be more complex than the C-fiber GABAergic pathway.     

Dependence of calcium influx in neocortical cells on temporal structure of depolarization, number of spikes, and blockade of NMDA receptors

Increase of intracellular [Ca(2+)] evoked by action potentials in a cell can induce long-term synaptic plasticity even without concomitant presynaptic stimulation. We used optical recording of the fluorescence of a Ca(2+)-indicator Oregon Green to investigate whether differences in results obtained with modifications of that purely postsynaptic induction protocol could be due to differential Ca(2+) influx. We compared changes of the somatic [Ca(2+)] in layer II-III pyramidal cells in slices of rat visual cortex evoked by bursts of depolarization pulses and long depolarizing steps. During weak depolarizations, the Ca(2+) influx was proportional to the amplitude and duration of the depolarization. With suprathreshold depolarizations, the Ca(2+) influx was proportional to the number of action potentials. Because the burst depolarizations evoked more spikes than did the long duration steps, this burst protocol led to a larger Ca(2+) influx. With all stimulation protocols, the spike-induced Ca(2+) influx was reduced during blockade of N-methyl-D-aspartate (NMDA) receptors. Differences in intracellular [Ca(2+)] increases thus may be one reason for differential effects of purely postsynaptic challenges on synaptic transmission.     

Deafferentation Weakens Excitatory Synapses in the Developing Central Auditory System

Decreased excitatory synaptic activity during development often leads to pre- and postsynaptic atrophy, as assessed anatomically. The present study considers the effect of decreased excitatory transmission on the maturation of synaptic strength. Towards this end, cochlear nucleus neurons, which project to the ipsilateral lateral superior olive (LSO), were denervated in gerbils at postnatal day 7, before the onset of hearing. This manipulation was intended to disrupt spontaneous glutamatergic transmission in the LSO while sparing the glycinergic afferents from the medial nucleus of the trapezoid body (MNTB). Afferent-evoked synaptic activity was assessed 1–6 days after ablation in a brain slice preparation using whole-cell current- and voltage-clamp recordings. In control animals, ipsilaterally evoked excitatory postsynaptic potentials (EPSPs) were present in 91% of neurons tested, but were observed in only 60% of neurons following cochlea removal. The maximum EPSP amplitude was significantly smaller in manipulated neurons compared with controls, and this was accompanied by a higher incidence of ipsilaterally evoked inhibitory postsynaptic potentials (IPSPs). To study the efficacy of excitatory synapses in greater detail, voltage-clamp recordings were made in the presence of strychnine and AP-5 [d(O)-2-amino-5-phosphonopentanoic acid]. The minimum excitatory postsynaptic current (EPSC) amplitude, presumed to reflect the efficacy of a single glutamatergic afferent, was ～40% smaller in manipulated neurons. In contrast, MNTB-evoked IPSPs were similar in neurons from control and ablated animals. However, manipulated neurons often exhibited a rebound depolarization after a hyperpolarizing current pulse or an afferent-evoked IPSP. In 70% of manipulated neurons, synaptically evoked rebound depolarizations were reduced, but not eliminated, by glutamate receptor antagonists. The glycine receptor antagonist strychnine did eliminate the IPSP-associated depolarization in these neurons. Collectively, these results suggest that functional denervation of excitatory afferents decreases their synaptic efficacy as result of both cell loss as well as decreased strength of individual surviving synapses.     

Dysfunction of small myelinated afferents in diabetic polyneuropathy, as assessed by laser evoked potentials.

OBJECTIVE: To verify whether laser evoked potentials are useful in assessing the function of small afferent fibers and to compare dysfunction of large and small afferent fibers in patients with diabetic polyneuropathy. METHODS: The brain potentials evoked by CO2 laser stimulation of the hand and foot were studied in diabetic patients (n = 45) with various degrees of peripheral nerve damage. Laser evoked potentials (which assess the function of small myelinated afferents) were also compared with ulnar and sural nerve sensory action potentials (which assess the function of large myelinated afferents) by scoring the abnormalities of the two neurophysiological tests with similar criteria. RESULTS: Laser evoked potentials were often absent; the mean latency was normal and mean amplitude decreased, as expected in axonopathies. Although clinical examination showed more frequent impairment of vibratory than pinprick sensation, laser evoked potentials and sensory action potentials yielded similar abnormality scores and showed a strong intra-individual correlation. CONCLUSIONS: Laser evoked potentials, possibly better than standard clinical examination for assessing the abnormalities of small-diameter afferents, indicate that diabetic polyneuropathy induces large- and small-afferent dysfunction in parallel.     

Enkephalins modulate excitatory synaptic transmission in the superficial dorsal horn by acting at μ-opioid receptor sites

The effect of opioids on synaptic potentials of dorsal horn (DH) neurons has been investigated in a rat spinal cord DH slice-dorsal root ganglion (DRG) in vitro preparation. Conventional intracellular recording from DH and DRG neurons using 3 M potassium acetate-filled electrodes was employed. Dorsal roots were electrically isolated from the spinal cord slice and stimulated with pulses of different intensity and duration to evoke afferent action potentials monitored intracellularly from DRG neurons. Low-intensity single-shock stimulation of the dorsal roots (8–20 V pulses of 0.02–0.05 ms duration) activated large primary afferents and elicited excitatory postsynaptic potentials (EPSP) in all of the neurons tested. High-intensity stimulation of the dorsal roots (over 35 V pulses of 0.5 ms duration), sufficient to excite small myelinated and unmyelinated primary afferents resulted in a large and prolonged depolarization of DH neurons associated with firing of action potentials. Bath application (d-Ala², N-Me-Phe⁴,Gly⁵-ol)-enkephalin (DAGO), (d-Ala², d-Leu⁵)-enkephalinamide (DADLEA), or (d-Ala², d-Met⁵)-enkephalinamide (DADMEA) produced dose-dependent, reversible hyperpolarization in about 75% of the neurons tested. The hyperpolarization was associated with a fall in neuronal input resistance. In addition, opioids depressed the synaptic transmission in all of the neurons examined. This depressant effect of opioids was independent from their effects on resting membrane potential. Delta specific receptor opioid agonists (d-Pen^2.5)-enkephalin (DPDPE) and (d-Pen², l-Pen⁵)-enkephalin (DPLPE), were completely ineffective in producing an effect on neuronal membrane or synaptic transmission. All opioid effects were antagonized by naloxone.     

Persistence of PAD and presynaptic inhibition of muscle spindle afferents after peripheral nerve crush

Two to twelve weeks after crushing a muscle nerve, still before the damaged afferents reinnervate the muscle receptors, conditioning stimulation of group I fibers from flexor muscles depolarizes the damaged afferents [M. Enriquez, I. Jimenez, P. Rudomin, Changes in PAD patterns of group I muscle afferents after a peripheral nerve crush. Exp. Brain Res., 107 (1996), 405-420]. It is not known, however, if this primary afferent depolarization (PAD) is indeed related to presynaptic inhibition. We now show in the cat that 2-12 weeks after crushing the medial gastrocnemius nerve (MG), conditioning stimulation of group I fibers from flexors increases the excitability of the intraspinal terminals of both the intact lateral gastrocnemius plus soleus (LGS) and of the previously damaged MG fibers ending in the motor pool, because of PAD. The PAD is associated with the depression of the pre- and postsynaptic components of the extracellular field potentials (EFPs) evoked in the motor pool by stimulation of either the intact LGS or of the previously damaged MG nerves. These observations indicate, in contrast to what has been reported for crushed cutaneous afferents [K.W. Horch, J.W. Lisney, Changes in primary afferent depolarization of sensory neurones during peripheral nerve regeneration in the cat, J. Physiol., 313 (1981), 287-299], that shortly after damaging their peripheral axons, the synaptic efficacy of group I spindle afferents remains under central control. Presynaptic inhibitory mechanisms could be utilized to adjust the central actions of muscle afferents not fully recovered from peripheral lesions.     

Locomotor-Related Presynaptic Modulation of Primary Afferents in the Lamprey

Presynaptic modulation of sensory afferent transmission during rhythmic motor activity was investigated in the lamprey spinal cord in vitro. Intracellular recordings were performed from the somata and axons of the glutamatergic sensory neurons from the skin (dorsal cells) during locomotor activity induced by N-methyl-D-aspartate (NMDA). Dorsal cells were phasically depolarized during each ipsilateral ventral root burst. In some soma recordings no or only small amplitude depolarizations were seen, although intracellular recording of their axons revealed the existence of large depolarizations, suggesting that the input synapses are located on the axons. The amplitude of the depolarizations increased during intracellular injection of hyperpolarizing current. The amplitude of the depolarizations increased when the frequency of the locomotor rhythm was increased by elevating the NMDA concentration. The depolarizations were not blocked by specific GABA_A (bicuculline) or GABA_B (phaclofen and saclofen) antagonists. To investigate whether the phasic depolarization may influence the monosynaptic excitatory transmission to giant interneurons, the amplitude of the monosynaptic excitatory postsynaptic potential (EPSP) was compared between the onset of the ipsilateral locomotor burst and the burst mid-point. The compound monosynaptic EPSP evoked from dorsal column was significantly smaller during the peak depolarization than at burst onset. The reduction of the amplitude of the EPSPs was not associated with any change of the membrane potential or input resistance of the giant interneurons, suggesting that this effect is mediated by a presynaptic mechanism. Phase-dependent effects were also seen on burst and cycle duration following dorsal column stimulation. Thus, the locomotor-related depolarizations in dorsal cell axons may represent a mechanism for a phasic gain control of sensory transmission during fictive locomotion.     

Effects of Ruthenium ions on the sensory terminal discharges of the frog muscle spindle

The presence of a mixed Na+-Ca2+ spike along the sensory terminal of the frog muscle spindle was verified. When the terminal was perfused with Ringer's solution containing 0.1-0.5 mM ruthenium red (RuR), the amplitude and duration of the spike were increased, occurring as a prolonged or a long-lasting depolarization of up to 20-30 s duration following individual afferent spikes evoked spontaneously or antidromically by electrical stimulation. In an isotonic TEA solution, the amplitude and duration of the afferent spikes were increased; however, no prolonged depolarization occurred. Adding 0.2 mM RuR to the TEA solution produced the prolonged and long-lasting depolarization. All responses disappeared in the presence of 3 microM TTX or Na+-free Ringer's solution. An impedance decrease along the terminal was observed during the prolonged or long-lasting depolarization. The prolonged depolarization was blocked by the addition of Ca2+-blockers; the afferent spikes remained. In preparations preincubated with 0.1 mM RuR, increasing CaCl2 in Ringer's solution from 0.2 mM, resulted in shortening of the duration of individual spikes with prolonged depolarization and in increase in the maximum rate of rise (MRR) of the spikes. Preincubation with higher concentrations of RuR produced higher sensitivities in the modifications of the duration and MRR to the change in [Ca2+]O. The responses were retained by adding RuR or RuCl3 to Ca2+-free Ringer's solution containing 0.1-5 mM EGTA, although all responses disappeared in Ca2+-free EGTA Ringer's solution. It is concluded that the RuR-induced prolonged response is produced by an influx of Na+.     

Modifications of afferent activities from Tibialis anterior muscle in rat by tendon vibrations, increase of interstitial potassium or lactate concentration and electrically-induced fatigue

Although previous experiments with a partially similar objective have been described in dogs, cats and rabbits, the purpose of this study was to identify and characterize mechanosensitive and chemosensitive muscle afferents in the anaesthetized rat since it is a widely used laboratory animal. The peroneal nerve innervating the tibialis anterior muscle was studied. Measurement of conduction velocities from compound action nerve potentials evoked by peripheral nerve stimulation allowed identification of group I-II (10.79+/-1.02 m/s), group III (2.96+/-0.58 m/s) and group IV (0.46+/-0.07 m/s) afferent fibers. Computation of the different compound potential areas showed that afferents I and II arising from spindles and tendon organs represented 9.65+/-2.2%, whereas afferents III and IV arising from free nerve endings in muscle represented 90.35+/-2.2% (III, 46.66+/-2.71% and IV, 43.69+/-2.52%). Action potentials were recorded from teased nerve filaments. Mechanical tendon vibrations (10 to 90 Hz) were used to activate mechanoreceptors. Peak increase in afferent discharge (fimpulses) was measured at 50 Hz (n = 12/19 units) or 70 Hz (n = 7/19 units). Intra-arterial bolus injections of different concentrations of potassium chloride (KCl: 1 to 20 mM) or lactic acid (LA: 0.5 to 3 mM) elicited marked activation of III and IV afferents (n = 124). Enhancement of fimpulses was not proportional to the increase in [KCl] or [LA]. Activation of afferents plateaued when [KCl] was equal or greater than 5 mM while fimpulses peaked, then decreased, when [LA] was 1 mM. Muscle fatigue induced by direct electrical muscle stimulation (EIF) markedly activated group III-IV (n = 17/18) afferents (176.9+/-29.7% of control) which persisted for the 3 minutes of recovery from fatigue. Maximal fimpulses increases in response to LA (+67%) and KCl (+46.9%) injections and to EIF (+76.9%) were similar. This procedure for characterizing the functional properties of sensory nerve endings in a skeletal muscle may be used to assess further changes in sensory muscle paths in experimental rodent pathophysiological systems.     

Reflex patterns in preganglionic sympathetic neurons projecting to the superior cervical ganglion in the rat

Reflex patterns in preganglionic neurons projecting in the cervical sympathetic trunk (CST) were analyzed in response to stimulation of various afferent systems. We focused on the question whether these preganglionic neurons can be classified into functionally distinct subpopulations. Reflex responses were elicited by stimulation of trigeminal and spinal nociceptive, thermoreceptive as well as baroreceptor and chemoreceptor afferents. Multi- and single fiber preparations were studied in baroreceptor intact and sino-aortically denervated animals. Spontaneous activity of 36 preganglionic single neurons ranged from 0.2 to 3.5 imp/s (median= 1.11 imp/s). The degree of cardiac rhythmicity (CR) in the activity of sympathetic neurons was 69.5+/-13% (mean+/-S.D.; N=52; range=39-95%). Noxious stimulation of acral skin activated the majority (67%) of sympathetic preparations by 37+/-25% (N=35) above pre-stimulus activity; 15% were inhibited. In these neurons the response to noxious stimulation of acral skin was significantly correlated with the degree of CR (P<0.001, N=52) in that neurons showing the strongest excitation to noxious stimulation displayed the strongest CR. Noxious mechanical stimulation of body trunk skin (N=60) inhibited the majority (80%) of fiber preparations tested (by 34+/-18% of pre-stimulus activity, N=48); an activation was not observed. Cold stimulation of acral (N=9) and body trunk skin (N=42) activated most fiber preparations. Trigeminal stimulation evoked a uniform reflex activation of preganglionic neurons (+79+/-73% of pre-stimulus activity, N=32). Chemoreceptor stimulation by systemic hypercapnia elicited inhibitory (-31+/-19%, N=8) as well as excitatory (+59+/-5%, N=4) responses. These results show that preganglionic sympathetic neurons projecting to target organs in the head exhibit distinct reflex patterns to stimulation of various afferent systems; however, a clear classification into different functional subgroups did not emerge. Furthermore, reflex patterns showed a segmental organization to noxious cutaneous stimulation of acral parts and body trunk reflecting a differential central integration of spinal afferent input. Compared with the cat the reflex organization of sympathetic neurons projecting to the head seems to be less differentiated in the anesthetized rat.     

Role of unmyelinated fibers in electroacupuncture cardiovascular responses

The afferent fiber type responsible for the transmission of sensory neural traffic to the central nervous system during acupoint stimulation is uncertain. Several early studies evaluating compound action potentials have suggested that myelinated fibers contribute to the afferent input of the autonomic reflex adjustments during electroacupuncture (EA). Our more recent data, employing single unit recordings of somatic afferents, show that both myelinated and unmyelinated fibers are stimulated by EA, although more finely myelinated than unmyelinated fibers are activated by low frequency, low current stimulation. We hypothesized in this study that unmyelinated group VI fibers also contribute significantly to the inhibitory influence of EA on cardiovascular pressor responses. We found that neonatal capsaicin-treated rats depleted of substance P from primary afferents were insensitive to the inhibitory EA effect during gastric distention. Thus, EA at P5-P6 reduced gastric distention-induced pressor responses from 19+/-3 to 11+/-2 mmHg in eight untreated rats while capsaicin-treated rats (n=9) were unresponsive to EA. Substance P containing neurons in dorsal root ganglion cells at Ti-T5 were significantly decreased in the capsaicin-treated rats from 47+/-4 to 22+/-4 cells. Treated compared to untreated rats also demonstrated a significantly (P<0.03) reduced number of group IV fibers identified with single unit recording techniques. This study demonstrates that the inhibitory effect of EA at P5-P6 on cardiovascular autonomic excitatory reflexes involves unmyelinated group IV fibers of the median nerves.     

Electrical properties and synaptic potentials of rabbit pancreatic neurons

Pancreatic ganglia receive innervation from a wide variety of extrinsic nerves and supply the predominant innervation to pancreatic acini, islets, and ducts. This study used intracellular recordings to investigate the electrical properties and synaptic potentials of rabbit pancreatic neurons. Neurons had a mean resting membrane potential of -54+/-0.4 mV and generated action potentials with a mean overshoot of 10+/-0.4 mV and a mean after-spike hyperpolarization (ASH) of 11+/-0.5 mV with duration of 210+/-19 ms. Action potentials exhibited a high threshold (-15+/-1 mV) for intracellular stimulation and a phasic firing pattern was observed in response to prolonged depolarizing currents. Stimulation of attached nerve bundles evoked multiple fast excitatory postsynaptic potentials (fEPSPs) which were abolished by hexamethonium in 75% of neurons, while a non-cholinergic fEPSP was observed in 25% of the neurons. Repetitive stimulation (3-30 Hz) evoked muscarinic slow EPSPs with a mean amplitude of 8+/-2 mV and duration of 5+/-1 s in a small subset (21%) of neurons. Exogenous muscarine evoked a mean slow depolarization of 10+/-1 mV amplitude in 22% of neurons tested. Following repetitive nerve stimulation non-cholinergic late, slow EPSPs with a mean amplitude of 4.3+/-0.4 mV were recorded in 32% of neurons. Nicotinic transmission was subject to inhibition mediated by presynaptic muscarinic receptors at low (0.5 Hz) stimulus frequencies in 80% of neurons. At higher frequencies (> or =1 Hz), either facilitation or depression of nicotinic transmission was observed depending on the ganglion studied. A population (9%) of neurons exhibited spontaneous, low-amplitude pacemaker-like potentials. Spontaneous fEPSPs and action potentials were also observed and these occasionally occurred in rhythmically timed bursts. Thus, distinct subpopulations of pancreatic neurons could be identified on the basis of both their intrinsic electrical properties and the receptors mediating and/or modulating synaptic transmission. These neurons function as critical sites of integration for synaptic input from extrinsic pancreatic nerves and thereby determine the postganglionic firing patterns presented to the pancreatic exocrine and endocrine secretory cells.     

Importance of neurokinin‐1 receptors in the nucleus tractus solitarii of mice for the integration of cardiac vagal inputs

Unmyelinated vagal afferents from the heart terminate within the nucleus tractus solitarii (NTS) located in the dorsomedial medulla. The neurotransmitter and postsynaptic receptors mediating information from cardiac vagal receptors to the NTS are unknown. This study determined the effects of neurokinin-1 (NK₁) receptor blockade on: (i) the reflex response evoked following aortic root injection of either veratridine (1–3 μg/kg) or bradykinin (80–300 ng/kg) to stimulate cardiac receptors in in vivo anaesthetized mice; and (ii) the evoked synaptic response of cardioreceptive NTS neurons following both intraleft-ventricular injection of veratridine or bradykinin, and electrical stimulation of the ipsilateral vagus nerve in an arterially perfused working heart-brainstem preparation of mouse. Administration of CP-99,994 (0.75–1.5 mg/kg i.v.), a specific NK₁ antagonist, attenuated significantly the evoked reflex bradycardia and depressor response following cardiac receptor (n = 6), but not pulmonary chemoreflex stimulation in vivo. From extracellular recordings of cardioreceptive NTS neurons, CP-99,994 reduced reversibly the total number of evoked spikes, peak firing frequency and response duration evoked by intraventricular injections of veratridine (n = 5) or bradykinin (n = 5). The number of evoked action potentials following electrical stimulation of the vagus nerve was also reduced. In five whole cell recordings of NTS neurons, both the evoked depolarization following cardiac receptor stimulation, and the peak amplitude and duration of vagus nerve-evoked EPSPs were reduced by CP-99 994; synaptic inputs from both peripheral chemoreceptors or pulmonary C-fibres were unaffected. These data support a selective involvement of NK₁ receptors in the transmission of cardiac vagal afferent inputs to NTS neurons integrating cardiorespiratory information.     

Pharmacology of the vestibular hair cell-afferent fiber synapse in the frog

The isolated, intact, membranous labyrinth of the frog (Rana temporaria) has been investigated electrophysiologically in vitro to determine the nature of the transmitter substance at the synapse between the vestibular hair cells and afferent fibers. Spontaneous synaptic activity can be monitored with intra-axonal recordings from the afferents. Increased K+ in the bath results in an increase in frequency of presynaptic release, as indicated by an increased frequency of spontaneous synaptic potentials. Adding Mg2+ and lowering Ca2+ results in a decrease in synaptic potential frequency (often to zero) with no change in their mean amplitude, indicating pre-synaptic blockade. Extracellular recordings from individual vestibular afferents indicate that bath-applied glutamate and related acidic amino acids consistently increase the firing rates of these afferents in a dose-dependent manner with no evidence of desensitization. In the presence of presynaptic blockade (high Mg2+/low Ca2+), bath application of glutamate and its agonists results in a reversible depolarization of vestibular afferents, suggesting a postsynaptic action of these substances. 2-Amino-5-phosphonovaleric acid, kynurenic acid, and other acidic amino acid antagonists reversibly decrease the amplitudes of spontaneously occurring synaptic potentials without affecting their frequency, indicating subsynaptic blockade. These antagonists also block the postsynaptic depolarizations due to glutamate and its agonists. GABA and its agonists and antagonists have no consistent effect upon afferent activity. These findings suggest that glutamate, aspartate, or a related compound is the transmitter at this synapse. However, the antagonists used, or the receptors themselves, are not selective enough to discriminate adequately between the agonists. Therefore, which of these glutamate agonists are actually involved in synaptic transmission remains to be determined.     

The effect of selective electrical stimulation of non-myelinated vagal fibres on heart rate in the rabbit

The bradycardia evoked by electrical stimulation of the peripheral cut end of the rabbit vagus nerve is mediated by both myelinated and non-myelinated fibres. The purpose of this study was to assess the effects of non-myelinated fibres on heart rate in the rabbit using selective electrical stimulation techniques. In 8 rabbits selective activation of non-myelinated fibres using reversed polarity triangular shaped pulses (10 Hz, 20 s), resulted in a slowly developing fall in heart rate of 24.1 +/- 1.1 beats/min which outlasted the period of stimulation by 58.4 +/- 4.2 s. In 4 rabbits stimulation of myelinated fibres at 10 Hz for 20 s resulted in a fall in heart rate of 24.5 +/- 2.6 beats/min. On stimulation of both myelinated and non-myelinated fibres heart rate fell by 39.9 +/- 3.2 beats/min. Heart rate returned rapidly to control value following stimulation of myelinated fibres (5.6 +/- 0.5 s) but only slowly after stimulation of both myelinated and non-myelinated fibres (56.7 +/- 4.9 s). Atropine (5 mg/kg, i.v.) abolished all effects of vagal stimulation on heart rate. Hexamethonium (15 mg/kg, i.v.) abolished the effect of myelinated fibres on heart rate but did not affect the fall in heart rate produced by non-myelinated fibres. We suggest that the prolonged effects on stimulation of non-myelinated fibres may reflect the persistent action of a non-cholinergic excitatory transmitter at the cardiac parasympathetic ganglia.     

Presynaptic inhibition of identified wind-sensitive afferents in the cercal system of the locust

The paired cerci located at the tip of the locust abdomen bear a large number of wind-sensitive filiform hairs, each of which sends an axon via the cercal nerve into the terminal ganglion of the CNS. The filiform afferents fire bursts of action potentials when their hairs are displaced by wind or mechanical stimuli. Filiform axon terminals in the CNS are depolarized concomitantly with the discharge of another type of unit (a primary afferent-depolarizing, or PAD, unit) recorded in the cercal nerve. The instantaneous spike frequency of PAD unit discharges matches the evoked depolarization very closely, and during such depolarizations spike amplitudes in the filiform afferent terminals are reduced by up to 55%. Depolarizing current pulses injected into the axonal terminals of an identified filiform afferent evoke spikes that are blocked by the PAD unit, probably via an intercalated interneuron. The PAD unit makes a monosynaptic connection with only one of the 4 giant interneurons (GIN 2) on each side of the terminal ganglion, and indirect connections with 2 others. Depolarizing current pulses injected into the neuropilar segments of GINs evoke fewer spikes when the PAD unit is active, consistent with the PAD unit's mediation of conductance changes in postsynaptic cells. Iontophoretic injection of Lucifer yellow shows the PAD unit to be an afferent with axon terminals overlapping those of filiform afferents and posteriorly directed branches of interneurons such as GIN 2 in the CNS. Passive movements of a cercus, monitored with a position transducer, show that the PAD unit fires discrete bursts during cercal displacement. The PAD unit most probably has its soma and dendrites in tissue spanning the cercal base. By responding to cercal movements sufficient to also activate filiform hairs, and by mediating conductance changes in both the presynaptic terminals of filiform afferents and the postsynaptic membranes of interneurons, the PAD unit desensitizes a pathway to movement-generated afferent input, and ensures that the locust remains sensitive to external wind stimuli.     

Long-term potentiation of silent synapses in substantia gelatinosa neurons

In this study, we observed quantal changes in single synapse excitatory postsynaptic currents to characterize N-methyl-D-aspartate receptor-mediated silent synapses between primary afferents and spinal substantia gelatinosa neurons. The failure rate of primary afferent quantal excitatory postsynaptic currents was lower at depolarized holding potentials than at hyperpolarized potentials. This lower failure rate at depolarized potentials was due to the activation of N-methyl-D-aspartate receptor-mediated excitatory postsynaptic currents. Pairing primary afferents with the postsynaptic depolarization induced a long-term decrease in the failure rate of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor-mediated excitatory postsynaptic currents, but did not alter the failure rate of N-methyl-D-aspartate receptor-mediated excitatory postsynaptic currents. Our findings suggest that pairing primary afferent with postsynaptic depolarization can convert silent substantia gelatinosa synapses to active synapses and the mechanism of this synaptic plasticity is associated with postsynaptic modification.     

Intersegmental connections and interactions of myelinated somatic and visceral afferents with sympathetic preganglionic neurons in the unanesthetized spinal cat

The aim of this study was to obtain a measure of the interactions through exclusively spinal circuits, of myelinated afferents with sympathetic preganglionic neurons. Experiments were performed on 16 unanesthetized cats rendered insensitive by bilateral vertebral and carotid occlusion, whose spinal cords had been transected at C1 6-12 h before recording. The evoked responses of 68 tonically active sympathetic preganglionic neurons were recorded from filaments dissected from the cervical sympathetic trunk. Excitation, inhibition and excitation-inhibition sequences were evoked by electrical stimulation of radial, femoral and pelvic nerve afferents. Inhibition was most often observed during pelvic nerve stimulation. Ninety percent of the sympathetic preganglionic neurons tested responded to radial, 77% to femoral and 85% to pelvic nerve stimulation. These differences in percentage of units responding to the three nerves were statistically insignificant. Thus, in the acute spinal cat, the fraction of tonically active sympathetic preganglionic neurons whose activity can be influenced by myelinated afferents is independent of the length of the intraspinal pathway which conveys the input. A main difference between the long pathway (mediating the responses to femoral or pelvic nerve) and the short pathway (mediating the responses to radial nerve) seems to be the efficacy of their connections. While single shocks reliably evoked responses from the radial nerve, trains (200 Hz, 20 msec) were usually necessary to elicit responses from femoral or pelvic nerve, indicating a requirement for summation in the long pathway.