Peter D. Brodfuehrer

Identification of Neural Circuits by Imaging Coherent Electrical Activity with FRET-Based Dyes

We show that neurons that underlie rhythmic patterns of electrical output may be identified by optical imaging and frequency-domain analysis. Our contrast agent is a two-component dye system in which changes in membrane potential modulate the relative emission between a pair of fluorophores. We demonstrate our methods with the circuit responsible for fictive swimming in the isolated leech nerve cord. The output of a motor neuron provides a reference signal for the phase-sensitive detection of changes in fluorescence from individual neurons in a ganglion. We identify known and possibly novel neurons that participate in the swim rhythm and determine their phases within a cycle. A variant of this approach is used to identify the postsynaptic followers of intracellularly stimulated neurons.

Initiation of swimming activity by trigger neurons in the leech subesophageal ganglion

Summary1.The aim of this study was to identify neurons in the subesophageal ganglion of the medicinal leech which initiate swimming activity and to determine their output connections. We found two bilaterally symmetrical pairs of interneurons, Tr1 and Tr2, located in the first division of the subesophageal ganglion which initiate swimming activity in the Isolated nervous system when depolarized with brief (1–3 s) current pulses. Tr1 and Tr2 are considered trigger neurons because elicited swimming episodes outlast the stimulus duration, and because the length of elicited swim episodes is nearly independent of the intensity with which Tr1 and Tr2 are stimulated.2.Tr1 and Tr2 have similar morphologies. The neurites of both cells cross contralaterally in the subesophageal ganglion, project posteriorly, and exit the subesophageal ganglion in the contralateral connective. The axons of Tr1 and Tr2 extend as far posterior as segmental ganglion 18 of the ventral nerve cord.3.Tr1 provides direct excitatory drive to three groups of segmental neurons which are capable of initiating swimming: swim-initiating interneurons (cells 204 and 205), serotonin-containing interneurons (cells 61 and 21), and the serotonergic Retzius cells. In addition, all Retzius cells in the subesophageal ganglion are excited directly by Tr1. These three groups of neurons are excited even if Tr1 stimulation is subthreshold for swim initiation.4.In contrast to Tr1, Tr2 stimulation evokes transient inhibition in swim-initiating and serotonin-containing interneurons, and has little immediate effect on Retzius cells. In addition, Tr2 indirectly inhibits several oscillator neurons, including cells 208, 33, and 60.5.When Tr1 is stimulated during a swimming episode the swim period decreases for several cycles, while stimulation of Tr2 during swimming episodes reliably resets the ongoing swimming rhythm.6.Our findings indicate that Tr1 and Tr2 are trigger neurons which initiate swimming activity by different pathways. These neurons also have functional interactions with the swim oscillator network since either Tr1 or Tr2 stimulation during swimming can modulate the ongoing swimming rhythm.

From Stimulation to Undulation: A Neuronal Pathway for the Control of Swimming in the Leech

Initiation and performance of the swimming movement in the leech (Hirudo medicinalis) are controlled by neurons organized at at least four functional levels—sensory neurons, gating neurons, oscillator neurons, and motor neurons. A paired neuron, designated as Trl, in the subesophageal ganglion of the leech has now been shown to define a fifth level, interposed between sensory and gating neurons. Cell Trl is activated by pressure and nociceptive mechanosensory neurons, which mediate bodywall stimulus-evoked swimming activity in intact leeches. In the isolated leech nervous system, brief stimulation of cell Trl elicits sustained activation of the gating neurons and triggers the onset of swimmning activity. The synaptic interactions between all five levels of control are direct. Discovery of the Trl cells thus completes the identification of a synaptic pathway by which mechanosensory stimulation leads to the swimming movements of the leech.

Developmentally-regulated sodium channel subunits are differentially sensitive to α-cyano containing pyrethroids

Juvenile rats have been reported to be more sensitive to the acute neurotoxic effects of the pyrethroid deltamethrin than adults. While toxicokinetic differences between juveniles and adults are documented, toxicodynamic differences have not been examined. Voltage-gated sodium channels, the primary targets of pyrethroids, are comprised of alpha and beta subunits, each of which have multiple isoforms that are expressed in a developmentally-regulated manner. To begin to test whether toxicodynamic differences could contribute to age-dependent deltamethrin toxicity, deltamethrin effects were examined on sodium currents in Xenopus laevis oocytes injected with different combinations of rat alpha (Na(v)1.2 or Na(v)1.3) and beta (beta(1) or beta(3)) subunits. Deltamethrin induced tail currents in all isoform combinations and increased the percent of modified channels in a concentration-dependent manner. Effects of deltamethrin were dependent on subunit combination; Na(v)1.3-containing channels were modified to a greater extent than were Na(v)1.2-containing channels. In the presence of a beta subunit, deltamethrin effects were significantly greater, an effect most pronounced for Na(v)1.3 channels; Na(v)1.3/beta(3) channels were more sensitive to deltamethrin than Na(v)1.2/beta(1) channels. Na(v)1.3/beta(3) channels are expressed embryonically, while the Na(v)1.2 and beta(1) subunits predominate in adults, supporting the hypothesis for age-dependent toxicodynamic differences. Structure-activity relationships for sensitivity of these subunit combinations were examined for other pyrethroids. Permethrin and tetramethrin did not modify currents mediated by either subunit combination. Cypermethrin, beta-cyfluthrin, esfenvalerate and fenpropathrin all modified sodium channel function; effects were significantly greater on Na(v)1.3/beta(3) than on Na(v)1.2/beta(1) channels. These data demonstrate a greater sensitivity of Na(v)1.3 vs Na(v)1.2 channels to deltamethrin and other cyano-containing pyrethroids, particularly in the presence of a beta subunit.

Ultrasound sensitive neurons in the cricket brain

Summary1.The aim of this study was to identify neurons in the brain of the cricket, Teleogryllus oceanicus, that are tuned to high frequencies and to determine if these neurons are involved in the pathway controlling negative phonotaxis. In this paper we describe, both morphologically and physiologically, 20 neurons in the cricket brain which are preferentially tuned to high frequencies.2.These neurons can be divided into two morphological classes: descending brain interneurons (DBINs) which have a posteriorly projecting axon in the circumesophageal connective and local brain neurons (LBNs) whose processes reside entirely within the brain. All the DBINs and LBNs have processes which project into one common area of the brain, the ventral brain region at the border of the protocerebrum and deutocerebrum. Some of the terminal arborizations of Int-1, an ascending ultrasound sensitive interneuron which initiates negative phonotaxis, also extend into this region.3.Physiologically, ultrasonic sound pulses produce 3 types of responses in the DBINs and LBNs. (1) Seven DBINs and 6 LBNs are excited by ultrasound. (2) Ongoing activity in one DBIN and 5 LBNs is inhibited by ultrasound, and (3) one cell, (LBN-ei), is either excited or inhibited by ultrasound depending on the direction of the stimulus.4.Many of the response properties of both the DBINs and LBNs to auditory stimuli are similar to those of Int-1. Specifically, the strength of the response, either excitation or inhibition, to 20 kHz sound pulses increases with increasing stimulus intensity, while the response latency generally decreases. Moreover, the thresholds to high frequencies are much lower than to low frequencies. These observations suggest that the DBINs and LBNs receive a majority of their auditory input from Int-1. However, the response latencies and directional sensitivity of only LBN-ei suggest that it is directly connected to Int-1.5.The response of only one identified brain neuron, DBIN8, which is inhibited by 20 kHz sound pulses, is facilitated during flight compared to its response at rest. This suggests that suppression of activity in DBIN8 may be associated with ultrasound-induced negative phonotactic steering responses in flying crickets. The other DBINs and LBNs identified in this paper may also play a role in negative phonotaxis, and possibly in other cricket auditory behaviors influenced by ultrasonic frequencies.

Kinematic and aerodynamic aspects of ultrasound-induced negative phonotaxis in flying Australian field crickets (Teleogryllus oceanicus)

SummaryNegative phonotaxis is elicited in flying Australian field crickets,Teleogryllus oceanicus, by ultrasonic stimuli. Using upright tethered flying crickets, we quantitatively examined several kinematic and aerodynamic factors which accompany ultrasound-induced negative phonotactic behavior. These factors included three kinematic effects (hindwing wingbeat frequency, hindwing elevation and depression, and forewing tilt) and two aerodynamic effects (pitch and roll).1.Within two cycles following a 20 dB suprathreshold ultrasonic stimulus, the hindwing wingbeat frequency increases by 3–4 Hz and outlasts the duration of the stimulus. Moreover, the relationship between the maximum increase in wingbeat frequency and stimulus intensity is a twostage response. At lower suprathreshold intensities the maximum wingbeat frequency increases by approximately 1 Hz; but, at higher intensities, the maximum increase is 3–4 Hz.2.The maximum hindwing elevation angle increases on the side ipsilateral to the stimulus, while there was no change in upstroke elevation on the side contralateral to the stimulus.3.An ultrasonic stimulus affects forewing tilt such that the forewings bank into the turn. The forewing ipsilateral to the stimulus tilts upward while the contralateral forewing tilts downward. Both the ipsilateral and contralateral forewing tilt change linearly with stimulus intensity.4.Flying crickets pitch downward when presented with a laterally located ultrasonic stimulus. Amputation experiments indicate that both the fore and hindwings contribute to changes in pitch but the pitch response in an intact cricket exceeds the simple addition of fore and hindwing contributions. With the speaker placed above or below the flying cricket, the change is downward or upward, respectively. For all cases, the magnitude of the pitch change is linearly related to stimulus intensity.5.Ultrasound induces flying crickets to roll away from the stimulus source location. The dynamics of the changes in roll are similar to the changes in pitch. That is, the change in roll is a function of both wing pairs and the magnitude is stimulus intensity dependent.6.Thus ultrasound-induced negative phonotaxis in flying crickets is a complex behavior involving several kinematic and aerodynamic changes. This behavioral complexity lends further credence to the hypothesis that cricket negative phonotaxis provides a valuable function, perhaps as a bat avoidance response.

The role of glutamate in swim initiation in the medicinal leech

Antagonists were used to investigate the role of the excitatory amino acid,l-glutamate, in the swim motor program ofHirudo medicinalis. In previous experiments, focal application ofl-glutamate or its non-NMDA agonists onto either the segmental swim-gating interneuron (cell 204) or the serotonergic Retzius cell resulted in prolonged excitation of the two cells and often in fictive swimming. Since brief stimulation of the subesophageal trigger interneuron (cell Tr1) evoked a similar response, we investigated the role of glutamate at these synapses. Kynurenic acid and two non-NMDA antagonists, 6,7-dinitroquinoxaline-2,3-dione (DNQX) and Joro spider toxin, effectively suppressed (1) the sustained activation of cell 204 and the Retzius cell following cell Tr1 stimulation and (2) the monosynaptic connection from cell Tr1 to cell 204 and the Retzius cell, but did not block spontaneous or DP nerve-activated swimming. Other glutamate blockers, including γ-d-glutamylaminomethyl sulfonic acid,l(+)-2-amino-3-phosphonoproprionic acid and 2-amino-5-phosphonopentanoic acid, were ineffective. DNQX also blocked both indirect excitation of cell 204 and direct depolarization of cell Tr1 in response to mechanosensory P cell stimulation. Our findings show the involvement of non-NMDA receptors in activating the swim motor program at two levels: (1) P cell input to cell Tr1 and (2) cell Tr1 input to cell 204, and reveal an essential role for glutamate in swim initiation via the cell Tr1 pathway.

Effect of the tail ganglion on swimming activity in the leech

In the medicinal leech, Hirudo medicinalis, isolated segmental nerve cords are capable of generating swimming activity. The role played by the head and tail ganglia in regulating the expression of swimming activity by the segmental nerve cord was evaluated by comparing swimming activity in nerve cord preparations with and without the head and tail ganglia attached. Several swim properties were examined, including length of induced swim episodes, ability to initiate swim episodes, swim cycle period, and phase. We found that, in general, the presence of the tail ganglion attached to isolated nerve cords countered the effects produced by the head ganglion on swimming activity. Moreover, we observed that the tail ganglion itself provides excitatory drive to the swim generating system. Thus, the inputs from the head and tail ganglia influence significantly the expression of swimming activity.

Activation of two forms of locomotion by a previously identified trigger interneuron for swimming in the medicinal leech

Higher-order projection interneurons that function in more than one behavior have been identified in a number of preparations. In this study, we document that stimulation of cell Tr1, a previously identified trigger interneuron for swimming in the medicinal leech, can also elicit the motor program for crawling in isolated nerve cords. We also show that motor choice is independent of the firing frequency of Tr1 and amount of spiking activity recorded extracellularly at three locations along the ventral nerve cord prior to Tr1 stimulation. On the other hand, during Tr1 stimulation there is a significant difference in the amount of activity elicited in the ventral nerve cord that correlates with the motor program activated. On average, Tr1 stimulation trials that lead to crawling elicit greater amounts of activity than in trials that lead to swimming.

Glutamate receptor 5/6/7-like and glutamate transporter-1-like immunoreactivity in the leech central nervous system

Previous physiological and pharmacological evidence has suggested a neurotransmitter role for the excitatory amino acid glutamate in the leech central nervous system (CNS). In the present study, we sought to localize glutamate receptor (GluR) subunits (GluR 5/6/7, GluR 2/3 and N‐methyl‐D‐aspartate receptor 1 [NMDAR 1]) and a glutamate transporter subtype [GLT‐1] within the leech CNS using mono‐ and polyclonal antibodies. In whole‐mounted tissue, small cells of the outer capsule and putative microglia labeled with both GluR 5/6/7 and GluR 2/3 but not NMDAR 1 subunit antisera. In general, GluR 5/6/7‐like immunofluorescence was both more intense and more widespread than GluR 2/3‐like immunolabeling. Cryostat‐sectioned tissue revealed extensive GluR 5/6/7‐like immunoreactivity throughout the neuropil as well as labeling within a few neuronal somata. GLT‐1‐like immunoreactivity localized to the inner capsule, which is the interface between neuronal somata and the neuropil and is deeply invested by processes of neuropil glia. These results complement previous physiological and pharmacological findings indicating that the leech CNS possesses the cellular machinery to respond to glutamate and to transport glutamate from extracellular spaces. Together, they provide further evidence for glutamates role as a neurotransmitter within the leech CNS. J. Comp. Neurol. 405:334–344, 1999.