Fig 1.
Diagrams of crayfish brain and lateral protocerebrum.
A, Dorsal view of the crayfish brain indicating major deutocerebral regions (shading) and the neural structures within the optic cups. Parasol cells in the hemiellipsoid body of the lateral protocerebrum (LP) are major targets for projection neurons from the accessory lobes. Projection neuron axons run within the olfactory globular tract, a subdivision of the lateral protocerebral tract (From [44] with permission). B, Position of various morphological regions of the parasol cells within the lateral protocerebrum. A parasol cell is shown within the hemiellipsoid body/ medulla terminalis complex. Intracellular recordings were made from large dendritic branches within the hemiellipsoid body (HEB). The medulla terminalis (MT) constitutes the major structure within the lateral protocerebrum.
Fig 2.
Isolated crayfish brain set-up.
Diagram of the isolated crayfish head preparation used in the present study. The brain, B, is indicated within the head capsule. Cannulas perfuse saline through the median and lateral cephalic arterial systems of the head. The lateral protocerebrum is indicated at its normal position within the right eyestalk, with covering structures removed. (Modified from [45]; used with permission).
Fig 3.
Background synaptic activity in lobster and crayfish parasol cells.
A, spontaneous bursting during background activity in a parasol cell from Homarus americanus, recurring at approximately 4-second intervals. B, irregular, occasional spontaneous bursting during background activity in a parasol cell of P. clarkii imaged on a similar time base. Note pause in background activity following the bursts in A and B. C, repetitive bursting in the same parasol cell as in B in response to a light pulse to the ipsilateral compound eye, the duration of which is approximated by the time marker below the electrical trace.
Fig 4.
Parasol cell responses to hyperpolarizing current.
A1, voltage sag and post-hyperpolarizing discharge produced in a crayfish parasol cell by a 10 sec pulse of hyperpolarizing current during background activity in normal saline. A2, sag produced by an identical current injection in the same neuron after treating the preparation with 5 x 10−7 M TTX. B, response of a TTX-treated crayfish parasol cell to a series of 0.2 nA steps of hyperpolarizing current. The threshold for onset of the voltage sag in this neuron was approximately 25 mV hyperpolarized to the resting potential. C, effect of the addition of 10 mM CsCl to the perfusate saline in a TTX-treated parsol cell. The voltage sag and depolarizing overshoot present in the response to a five second rectangular pulse of hyperpolarizing current in normal saline were reduced or eliminated in the saline containing Cs+ ions, and the recovery to resting potential level from the hyperpolarization took approximately one second longer. Broken lines in all records indicate zero membrane potential.
Fig 5.
Responses of parasol cells to Cesium saline.
Effects of 10 mM CsCl on the background activity of a crayfish parasol cell. A, preparation perfused with normal saline prior to application of 10 mM CsCl saline. B, after 15' in saline with Cs + ions. The maximum levels achieved by the resting membrane potential increased by 6 mV to –76 mV, and the frequency of background bursts was reduced by approximately 50%. C, recovery in normal saline after 55', by which time the resting potential had fallen to –74 mV.
Fig 6.
Experimental protocol to reveal IA.
Stimulation paradigm used to reveal the presence of IA. A, response of a parasol cell to a 4-sec rectangular pulse of depolarizing current. After a period of no current injection, a two-second pulse of hyperpolarizing current (0.2 nA) was injected into the neuron (bottom traces), followed immediately by the previous depolarizing current injection. As discussed in the text, the hyperpolarizing prepulse delayed the onset of the spike response to the depolarization. B, treatment of the cell with 1 mM 4-AP reduced the prepulse delay and promoted bursting in the parasol cell. Zero membrane potential is indicated by the dashed lines.
Fig 7.
Latency to first spike electrical records.
A, response of a parasol cell to a 4-sec pulse of depolarizing current (bottom trace). B-D, identical depolarizing pulses were preceded by 2-sec pulses of hyperpolarization, generated by a current level of -0.34 nAmp. In B, the delay to the first spike from the depolarizing onset was clearly increased when compared to the delay in A. In C, the preparation was treated with 1mM 4-AP for 10 min prior to the current pulses, with a concomitant reduction in delay to first spike. D shows nearly complete recovery of the delay following one hour in normal saline. Time calibration, 100 msec.
Fig 8.
Frequency histograms of data from two cells documenting prepulse latencies with and without 4-AP.
Frequency histograms of delays to first spike in two parasol cells in response to depolarizing current steps without (A1, A2) and with (C1, C2) a preceding hyperpolarizing pulse. Data in A1, A2, C1 and C2 were recorded in normal saline; identical tests were run in the two cells (B1, B2 and D1, D2) while they were in saline + 1mM 4-AP, which significantly reduced the post-hyperpolarization delay to first spike.
Fig 9.
Latency to first spike data from a parasol cell under various conditions and in response to different levels of injected current.
Delays to initial spike, under various experimental conditions, in response to constant depolarizing current steps immediately following 2-sec hyperpolarizing current pulses of varying amplitudes. Each data point is the mean of 10 repetitions at that current intensity, +/- one standard error. Black squares, response profile in normal saline; red circles, saline containing 5 mM CsCl; blue triangles, responses in saline with 5 mM CsCl and 1mM 4-AP; magenta triangles, responses after 90 min recovery in normal saline.
Fig 10.
Increased spontaneous bursting in saline with 4-AP.
Effects of 4-AP upon background activity in two parasol cells. A1, background activity in normal saline. A2, spontaneous bursting (*) in the presence of 1 mM 4-AP. A3, recovery in normal saline. B1, different parasol cell in normal saline. B2, following exposure to saline plus 1mM 4-AP. Bursts indicated by (*). Recording situation was lost immediately following return to normal saline. Dashed lines indicate zero membrane potential.