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Notes: Abstract 1. Electrotonic and chemical synaptic potentials were measured as a function of frequency of presynaptic action potentials. Over the frequency range from 0.02 to 10 Hz, the electrotonic synaptic potential was constant, while the chemical synaptic potential decreased in magnitude. Above 10 Hz, both synaptic events decreased in magnitude consistent with filtering by the dendritic structures. 2. Electrotonic synaptic transfer functions from 0.5 to 100 Hz were measured for the I 1 reticulospinal Müller axon to spinal neuron electrotonic synaptic junction of the lamprey spinal cord using paired recordings from the pre-synaptic terminals and the post-synaptic neurons. In addition to this two-point synaptic transfer function, individual single point impedance functions of both the postsynaptic soma and the pre-synaptic axon terminal were measured. 3. The measured functions were interpreted with a computational model based on a three dimensional reconstruction of a Lucifer yellow filled motoneuron. Simulations of the model for a synaptic location of the I 1, synapse were consistent with the measured synaptic transfer functions. 4. Synaptic potentials were simulated for inputs on dendrites near the I 1 axon as well as distal dendritic regions. The high frequency filtering increased as the synaptic location was moved from the soma to the periphery, but the potential response on distal dendrites was larger than would have been predicted from the end of the equivalent cylinder of a Rall model that was used to fit soma impedance functions. 5. Electrotonic post-synaptic potentials were enhanced by the activation of a TTX-sensitive negative conductance. The algebraic addition of the increased negative conductance and all of the positive conductances led to a decreased net conductance, i.e. an increased impedance. Thus, the same synaptic current caused a larger potential response proportional to the neuronal impedance. Post-synaptic potentials computed from the transfer function data showed an enhancement with depolarization similar to that observed by direct measurement. 6. Thus, measurements of point and transfer impedances of central neurons, coupled with simulations allow a quantitative description of the cable properties of dendritic processes including both passive filtering and active voltagedependent properties that may enhance synaptic potentials.

Notes: Abstract. Swimming in vertebrates such as eel and lamprey involves the coordination of alternating left and right activity in each segment. Forward swimming is achieved by a lag between the onset of activity in consecutive segments rostrocaudally along the spinal cord. The intersegmental phase lag is approximately 1% of the cycle duration per segment and is independent of the swimming frequency. Since the lamprey has approximately 100 spinal segments, at any given time one wave of activity is propagated along the body. Most previous simulations of intersegmental coordination in the lamprey have treated the cord as a chain of coupled oscillators or well-defined segments. Here a network model without segmental boundaries is described which can produce coordinated activity with a phase lag. This ‘continuous’ pattern-generating network is composed of a column of 420 excitatory interneurons (E1 to E420) and 300 inhibitory interneurons (C1 to C300) on each half of the simulated spinal cord. The interneurons are distributed evenly along the simulated spinal cord, and their connectivity is chosen to reflect the behavior of the intact animal and what is known about the length and strength of the synaptic connections. For example, E100 connects to all interneurons between E51 and E149, but at varying synaptic strengths, while E101 connects to all interneurons between E52 and E150. This unsegmented E-C network generates a motor pattern that is sampled by output elements similar to motoneurons (M cells), which are arranged along the cell column so that they receive input from seven E and five C interneurons. The M cells thus represent the summed excitatory and inhibitory input at different points along the simulated spinal cord and can be regarded as representing the ventral root output to the myotomes along the spinal cord. E and C interneurons have five simulated compartments and Hodgkin-Huxley based dynamics. The simulated network produces rhythmic output over a wide range of frequencies (1–11 Hz) with a phase lag constant over most of the length, with the exception of the ‘cut’ ends due to reduced synaptic input. As the inhibitory C interneurons in the simulation have more extensive caudal than rostral projections, the output of the simulation has positive phase lags, as occurs in forward swimming. However, unlike the biological network, phase lags in the simulation increase significantly with burst frequency, from 0.5% to 2.3% over the range of frequencies of the simulation. Local rostral or caudal increases in excitatory drive in the simulated network are sufficient to produce motor patterns with increased or decreased phase lags, respectively.

Notes: The lamprey spinal cord has been utilized to investigate the role of presynaptic inhibition in the control of the spinal motor system. Axons of the lamprey spinal cord are comparatively large because of their lack of myelination. Axons impaled with microelectrodes demonstrate depolarizing responses to the application of GABAA and GABAB receptor agonists, muscimol and baclofen. These depolarizing effects are counteracted by the specific GABAA and GABAB receptor antagonists, bicuculline and phaclofen. GABAA receptor activation leads to a gating of Cl− channels on the axons. However, the ionic mechanism leading to axonal depolarization following GABAB receptor activation is unknown. After initiation of fictive locomotion, these axons demonstrate oscillations in axonal membrane potential related to the locomotor cycle. During ficitive locomotion they depolarize in phase with the bursting of the ipsilateral ventral root of the same segment. These axonal membrane potential oscillations are due to a phasic GABAA and GABAB receptor-mediated gating of ion channels on the axonal membrane. Fictive locomotion in the lamprey spinal cord is largely unaffected by antagonism of one or other GABA receptor subtype alone, but is severely disrupted by simultaneous antagonism of both subtypes. In conclusions, we demonstrate, for the first time, an agonist-gated depolarization of a vertebrate presynaptic element measured by direct impalement of the axon under study. We also demonstrate that GABA-mediated presynaptic inhibition occurs in axons of spinal interneurons. It is not limited to the primary afferents as has previously been believed.

Notes: The effect of two nitric oxide (NO) donors, SIN-1 and DEA/NO, as well as of the inactive SIN-1 derivative molsidomin, was studied on locus coeruleus (LC) neurons in a slice preparation using intracellular recordings. In addition, the effect of the guanylate cyclase inhibitor ODQ was analysed. Furthermore, the effect of NO donors on cyclic guanosine monophosphate (GMP) levels in the LC was studied using the indirect immunofluorescence technique, and the expression of soluble guanylyl cyclase with in situ hybridization. In 36 of 66 LC neurons extracellular application of SIN-1 and DEA/NO caused a hyperpolarization and a decrease in apparent input resistance. In almost 20% of neurons SIN-1 increased the firing rate. No effect could be recorded with the brain-inactive SIN-1 derivative molsidomin. The membrane permeable cGMP analogue 8-bromo-cGMP imitated the action of SIN-1. The hyperpolarizing effect of SIN-1 and DEA/NO was attenuated by preincubation with the guanylyl cyclase inhibitor ODQ. The immunohistochemical analysis revealed lack of cGMP immunostaining in non-stimulated slices, whereas SIN-1 dramatically increased this staining in about 40% of the LC neurons, and these neurons were all tyrosine hydroxylase positive, that is noradrenergic. A large proportion of the LC neurons expressed soluble guanylyl cyclase mRNA. The present and previous results suggest that NO, released from a small number of non-noradrenergic neurons in the LC, mainly has an inhibitory influence on many noradrenergic neurons, by upregulating cGMP levels via stimulation of soluble guanylyl cyclase. As nitric oxide synthase is present only in a small number of non-noradrenergic neurons ( Xu et al. 1994 ), a few neurons may influence a large population of noradrenergic LC neurons, which in turn may control activity in many regions of the central nervous system.

Notes: In this study, the effects of exogenously applied and endogenously released dopamine (DA), a powerful modulator of the lamprey locomotor network, are examined on excitatory glutamatergic synaptic transmission between reticulospinal axons and spinal neurons. Bath application of DA (1–50 µm) reduced the amplitude of monosynaptic reticulospinal-evoked glutamatergic excitatory postsynaptic potentials (EPSPs). The effect of DA was blocked by the D2-receptor antagonist eticlopride, and mimicked by the selective D2-receptor agonist 2,10,11 trihydroxy-N-propyl-noraporphine hydrobromide (TNPA). Bath application of the DA reuptake blocker bupropion, which increases the extracellular level of dopamine, also reduced the monosynaptic EPSP amplitude. This effect was also blocked by the D2-receptor antagonist eticlopride. To investigate if the action of DA was exerted at the presynaptic level, the reticulospinal axon action potentials were prolonged by administering K+ channel antagonists while blocking l-type Ca2+ channels. A remaining Ca2+ component, mainly dependent on N and P/Q channels, was depressed by DA. When DA (25–50 µm) was applied in the presence of ω-conotoxin GVIA, a toxin specific for N-type Ca2+ channels, it failed to affect the monosynaptic EPSP amplitude. DA did not affect the response to extracellularly ejected d-glutamate, the postsynaptic membrane potential, or the electrical component of the EPSPs. DA thus acts at the presynaptic level to modulate reticulospinal transmission.

Notes: 5-HT is a potent modulator of locomotor activity in vertebrates. In the lamprey, 5-HT dramatically slows fictive swimming. At the neuronal level it reduces the postspike slow afterhyperpolarization (sAHP), which is due to apamin-sensitive Ca2+-dependent K+ channels (KCa). Indirect evidence in early experiments suggested that the sAHP reduction results from a direct action of 5-HT on KCa channels rather than an effect on the Ca2+ entry during the action potential [Wallén et al., (1989) J. Neurophysiol., 61, 759–768]. In view of the characterization of different subtypes of Ca2+ channels with very different properties, we now reinvestigate if there is a selective action of 5-HT on a Ca2+ channel subtype in dissociated spinal neurons in culture. 5-HT reduced Ca2+ currents from high voltage activated channels. N-type, but not L-type, Ca2+ channel blockers abolished this 5-HT-induced reduction. It was also confirmed that 5-HT depresses Ca2+ currents in neurons, including motoneurons, in the intact spinal cord. 8-OH-DPAT, a 5-HT1A receptor agonist, also inhibited Ca2+ currents in dissociated neurons. After incubation in pertussis toxin, to block Gi/o proteins, 5-HT did not reduce Ca2+ currents, further indicating that the effect is caused by an activation of 5-HT1A receptors. As N-type, but not L-type, Ca2+ channels are known to mediate the activation of KCa channels and presynaptic transmitter release at lamprey synapses, the effects of 5-HT reported here can contribute to a reduction in both actions.

Notes: Activity-dependent synaptic plasticity has been proposed as a contributory factor in the patterning of rhythmic network activity. However, its role has not been examined in detail. Here, paired or triple intracellular recordings have been made from identified neurons in the lamprey locomotor network to examine the potential relevance of activity-dependent synaptic plasticity in segmental and intersegmental spinal networks. Segmental inputs from glutamatergic excitatory interneurons (EIN) to ipsilateral glycinergic crossed caudal (CC) interneurons were facilitated or depressed during spike trains at 5–20 Hz. Connections between EINs were depressed. Glycinergic inputs from small ipsilateral inhibitory interneurons were depressed in motor neurons, but were facilitated in CC interneurons. Excitatory inputs from small crossing interneurons to motor neurons were depressed, whereas inhibitory inputs were unaffected. With the exception of connections between EINs, significant effects occurred with stimulation that mimicked interneuron spiking during network activity. Intersegmental EIN synaptic properties were also investigated. EIN inputs did not differ significantly when examined from zero to four segments rostral to motor neurons or CC interneurons. However, caudally located EINs evoked greater activity-dependent facilitation than did rostral EINs. Whilst the amplitude or plasticity of EIN inputs in the rostral or mid-trunk regions of the spinal cord did not differ, EINs in the caudal trunk region evoked greater facilitation. Synaptic transmission between locomotor network neurons thus exhibits activity-dependent plasticity in response to physiologically relevant stimulation. Activity-dependent plasticity could thus contribute to the patterning of the rhythmic network output.

Notes: We have examined the morphological relationship of neuropeptide Y (NPY) and GABAergic neurons in the lamprey spinal cord, and the physiological effects of NPY and GABAB receptor agonists on afferent synaptic transmission. NPY-containing fibres and cell bodies were identified in the dorsal root entry zone. NPY immunoreactive (–ir) fibres made close appositions with primary afferent axons. Co-localization of NPY and GABA-ir was found in the dorsal horn and dorsal column. Fifty-two per cent of NPY-ir profiles showed immunoreactivity to GABA at the ultrastructural level. Electron microscopic analysis showed that NPY-immunoreactivity was present throughout the axoplasm, including over dense core vesicles, whereas GABA-immunoreactivity was mainly found over small synaptic vesicles. Synthetic lamprey NPY, and the related peptide, peptide YY, reduced the amplitude of monosynaptic afferent EPSPs in spinobulbar neurons. NPY had no significant effect on the postsynaptic input resistance or membrane potential, the electrical component of the synaptic potential, or the response to glutamate, but it could reduce the duration of presynaptic action potentials, suggesting that it was acting presynaptically. NPY also reduced the excitability of the spinobulbar neurons, suggesting at least one postsynaptic effect. Because NPY and GABA colocalize, we compared the effects of NPY and the GABAB agonist baclofen. Both presynaptically reduced EPSP amplitudes, baclofen having a larger effect and a faster onset and recovery than NPY. The GABAB antagonist phaclofen reduced the effect of baclofen, but not that of NPY. We conclude that NPY and GABA are colocalized in terminals in the dorsal spinal cord of the lamprey, and that they have complementary actions in modulating sensory inputs.

Notes: Bath application of the tachykinin neuropeptide substance P (1 μm) for 10 min causes long-lasting (〉 24 h) modulation of the frequency and regularity of NMDA-evoked locomotor bursts in the lamprey. The change in burst frequency has an induction phase (〈 2 h), which depends on the potentiation of NMDA responses and an increase in intracellular calcium levels, and a maintenance phase (〉 2 h), that is blocked by translational protein synthesis inhibitors. Here, the maintenance phase has been examined further. Unlike translation inhibitors, the transcription inhibitors actinomycin D and 5,6-dichlorobenzimidazole riboside (DRB) failed to reverse the change in burst frequency 2–3 h after substance P application, suggesting that the protein synthesized at this time does not require de novo RNA synthesis. Transcription inhibitors, however, reversed the change in burst frequency 15–24 h after substance P application, as did brefeldin A, which disrupts the Golgi complex and thus interferes with the post-translational transport of proteins. The change in burst regularity was unaffected by transcription or translation inhibitors, but was partially reversed by protein kinase A inhibitors applied 2.5–8 h after substance P. The glycoprotein synthesis inhibitor 2-deoxygalactose did not affect the changes in burst frequency or burst regularity. These results suggest that there are two phases to the maintenance of the change in burst frequency: an intermediate protein-, but not RNA-, synthesis-dependent phase, and a final RNA-synthesis-dependent phase. The change in burst regularity is protein-synthesis-independent, but may depend on activation of protein kinase A for at least 8 h after substance P application.

Notes: We have examined the effects of the tachykinin substance P on the action potential of lamprey mechanosensory dorsal cells. Substance P increased the spike duration and reduced the afterhyperpolarization. These effects were mimicked by stimulation of the dorsal root, which contains tachykinin-like immunoreactive fibres. The tachykinin antagonist spantide II blocked the effects of both substance P and dorsal root stimulation. The spike broadening was voltage-dependent, and was due to the reduction of a 4-aminopyridine-sensitive potassium conductance. The spike broadening was mimicked by G-protein activators and blocked by the G-protein inhibitor GDPβS. Pertussis toxin did not block the effects of substance P. The spike broadening was blocked by the protein kinase C and CAMP-dependent protein kinase inhibitor H7, and by the specific protein kinase C antagonist chelerythrine, but not by the cAMP and cGMP-dependent protein kinase inhibitor H8. The phorbol ester phorbol 12,13-dibutyrate mimicked and blocked the effects of substance P, supporting the role of protein kinase C in the spike modulation. The adenylate cyclase activator forskolin and the cAMP agonist SpcAMPs mimicked but did not block the effects of substance P on the spike duration, suggesting that protein kinase A also modulates the dorsal cell action potential, but that substance P acts independently of this pathway. Substance P also increased the excitability of the dorsal cells. This effect was blocked by 4-AP, PDBu and chelerythrine, but not by H8, suggesting that the increase in excitability shares the same intracellular and effector pathways as the spike broadening.