Running head: RECORDINGS OF LEECH MECHANOSENSORY NEURONS 1

Intracellular Recordings of Leech Mechanosensory Neurons:

Mapping Receptive Fields and Determination of Receptor Subtypes
Mackenzie M. Andrews
University of Washington - Neurobiology
RECORDINGS OF LEECH MECHANOSENSORY NEURONS 2

Abstract
An organism’s survival is dependent on its ability to encode environmental stimuli into neural signals; a
process called sensory transduction. One type of signal is somatosensation which includes touch,
temperature, body position, and pain. In order to differentiate between these different classes of stimuli,
neurons are specialized to only transduce certain types of stimuli from finite spatial and temporal regions.
This stimulus response fingerprint is called a neuron’s receptive field. To investigate somatosensory
transduction, we recorded from the medicinal leech (Hirudo verbena) mechanosensory neurons while
stimulating the intact skin of the animal. We successfully recorded from each of the three types of
mechanosnesory neurons: touch, pressure, and nociception transducers. By delivering two different
intensities of stimulus across the skin, we created a receptive field map for each neuron. Our results
suggest that pressure neurons have a smaller spatial receptive field than touch and nociception neurons
and that the intensity of a stimulus is encoded by an increase in frequency response.
Keywords: sensory encoding, mechanoreceptors, receptive field
RECORDINGS OF LEECH MECHANOSENSORY NEURONS 3

Intracellular Recordings of Leech Mechanosensory Neurons:

Mapping Receptive Fields and Determination of Receptor Subtypes
Introduction
The nervous system is responsible for encoding environmental stimuli into neural signals. This
process is called sensory transduction. While the nervous system’s ability to transduce sensory stimuli is
capacious, individual sensory neurons are only capable of transducing across a finite region of space or
time. This multidimensional range of transduction capacity is referred to as a neuron’s receptive field.
The receptive field of a neuron includes the direct space onto which that neuron responds to sensory
stimulus, as well any spatial or temporal component that affects that neuron’s response (e.g. synapses
from other neurons responding directly to different spatial fields).
A neuron’s receptive field is often multi-dimensional, restricted by spatial and temporal limits as
well as stimuli type and intensity. These differences in stimulus types may be modal or submodal.
Examples of different receptor modalities include mechano-, thermo-, and chemoreceptors. Within each
modality are potential submodalities such as light touch, pain, and pressure for mechanoreception.
To investigate the receptive fields and submodal responses of sensory receptors, quantitative
recordings and qualitative observations were taken from the medicinal leech (Hirudo verbena)
mechanosensory neurons. The leech is a segmented worm with a nervous system made up of a chain of
ganglia. Each ganglion is composed of approximately 400 neurons with 14 mechanosensitive neurons.
Those 14 mechanosensory neurons are subdivided into three submodalites: 6 T-cells, 4 N-cells, and 4 P-
cells; responsible for encoding light touch, nociception, and pressure, respectively (Bosma et al., 2017).
T-cells and N-cells are both rapidly adapting and thus sense change in stimuli (Titlow et al, 2013). P-cells
on the other hand fire with continuous stimulation to encode the magnitude of a stimulus (Carlton &
McVean, 1995).
Intracellular recordings were taken from 3 different mechanosensory neurons in a single leech
ganglion. The recordings were taken while the surface of the intact leech skin was stimulated with either a
string or a dissecting probe. We attempted to record from each submodality of mechanosensitive neuron
according to the ganglion map from Muller et al., 1981. We hypothesized that the receptive field of the
neurons would be strongest at the 0-annulus with response decreasing symmetrically to stimuli in the
positive and negative directions. In addition, according to work by Francisco Fernández-de-Miguel, we
hypothesized that pressure neurons would have a smaller spatial receptive field than touch and
nociception neurons (De-Miguel, 1997).
RECORDINGS OF LEECH MECHANOSENSORY NEURONS 4

Methods
Animal Preparation
A leech was dissected such that a single ganglion was pinned out to the recording chamber with
the skin and body wall intact. The preparation included the ganglion as well as the dorsomedial, lateral
and ventromedial skin regions of the -7 to +8 annuli. A ground wire was placed into the leech ringer filled
bath. An intracellular recording electrode was then filled with 3 M KCl and attached to the
micromanipulator controlled head-stage to be inserted into the cell.
Wiring and Recording Setup
The recording electrode and ground wire were connected to intracellular amplifier to amplify the
signal. The signal was then processed through the Humbug to remove 60 Hz noise. The output was then
sent to the PowerLab box to be collected through LabChart and saved on the computer. The output of the
PowerLab was sent to the Stimulus Input of the intracellular amplifier to control the current input from
LabChart. The protocol “Intracellular_Setup” from the LabChart software was used for all recordings.
Data Collection and Analysis
Once the electrode was inserted into a mechanosensitive cell, a string was used to stimulate each
available annulus in the dorsomedial, lateral, and ventromedial regions of the skin on both the ipsi- and
contralateral sides of the body preparation (or until the electrode slipped out of the cell). Annuli were
labeled such that 0 was at the level of the ganglion, negative numbers were increasingly caudal and
positive numbers were increasingly rostral. The stimulation consisted of a brief down-up motion of the
stimulating tool. This stimulation technique was repeated with a dissecting probe. The responses were
recorded and commented in LabChart. This was repeated for 3 separate mechanosensitive cells. In the
second cell, adaptation data was taken by attaching the dissecting probe to a micromanipulator and
lowering the probe onto the skin of the animal, maintaining constant pressure for 17 seconds before lifting
the probe off of the skin. Each neuron was analyzed by recording its spike amplitude and half-width (time
a response held 50% or greater of its maximal amplitude). Responses were quantified by the number of
spikes and the frequency (calculated using the interspike innterval) directly after a stimulus was
presented. All statistics are represented as a Mean ± SD.
Results
Recording Locations
In order to test the difference in receptive field and response between the three submodalities of
mechanosensory neurons, we recorded from one of each type: P, N and, T (neurons labeled 1, 2, and 3,
respectively) in the locations shown in Figure 1.a.
RECORDINGS OF LEECH MECHANOSENSORY NEURONS 5

Neuron 1
Neuron 1 was predicted to be a pressure-sensitive neuron based on its location. The neuron had a
spike amplitude of 52.6 ± 0.4 mV, a half width of 4.0 ± 0.1 ms, and spontaneous firing rate of 0.54 ± 0.32
Hz.
Thread Stimulation
To determine the response of neuron 1 to light touch, we first stimulated the skin with a thread.
Thread stimulation in the dorsomedial and lateral columns didn’t elicit a change from spontaneous firing
rate (Figure 2.a). Thread stimulation in the ventromedial column elicited a response of 5 spikes at 22.41 ±
2.3 Hz and 2 spikes at 11.11 Hz at annulus 0 and 1, respectively (Figure 2.b).
Probe Stimulation
To determine the response of neuron 1 to a harder stimulus, we stimulated the skin with a
dissecting probe. Stimulation of ventromedial annuli -4, -3, and -2 elicited 10 spikes at 26.09 ± 2.60 Hz,
13 spikes at 25.81 ± 1.90 Hz, and 22 spikes at 24.10 ± 2.1 Hz, respectively (Figure 2.c). The electrode
then came out of the cell before more recordings could be made.
Neuron 2
Neuron 2 was predicted to be a nociceptive-sensitive neuron based on its location. The neuron
had a spike amplitude of 70.9 ± 0.2 mV, a half width of 3.4 ± 0.1 ms, and spontaneous firing rate of 0.18
± 0.08 Hz.
Thread Stimulation
To determine the response of neuron 2 to light touch, we stimulated the skin with a thread.
Thread stimulation didn’t cause the neuron to change from spontaneous firing rate (Figure 3.a).
Probe Stimulation
To determine the response of neuron 2 to a harder stimulus, we stimulated the skin with a
dissecting probe. Stimulation of the dorsomedial column at annuli -3, -2, 0, 1, 2, and 3 elicited 4 spikes at
16.30 ± 0.50 Hz, 1 spike, 5 spikes at 18.69 ± 0.42 Hz, 5 spikes at 11.87 ± 0.61 Hz, 3 spikes at 14.36 ±
0.23 Hz, and 2 spikes at 1.04 Hz (Figure 3.b). Spontaneous activity then increased to 1.70 ± 0.51 Hz.
Probe stimulation of the lateral wall at annuli 0, 1, 2, and 3 elicited 7 spikes at 18.05 ± 0.30 Hz, 9 spikes
at 17.89 ± 0.31 Hz, 5 spikes at 13.29 ± 0.45 Hz, and 3 spikes at 18.26 ± 0.42 Hz, respectively (Figure
3.b). Contralateral stimulation failed to evoke a response (Figure 3.c).
Prolonged Stimulation
A prolonged stimulation at the 0-annulus, lateral column, increased the spike frequency from the

spontaneous rate to 6.0 ± 0.1 Hz (Figure 4.a-b). The response frequency decreased throughout the

sustained stimulus.
RECORDINGS OF LEECH MECHANOSENSORY NEURONS 6

Neuron 3
Neuron 3 was predicted to be a touch-sensitive neuron based on its location. The neuron had a
spike amplitude of 33.6 ± 0.6 mV, a half width of 3.3 ± 0.1 ms, and spontaneous firing rate of 0.33 ± 0.05
Hz.
Thread Stimulation
To determine the response of neuron 3 to light touch, we stimulated the skin with a thread.
Dorsomedial stimulation resulted in 4 spikes at 15.11 ± 0.30 Hz, 4 spikes at 9.57 ± 0.41 Hz, and 10 spikes
at 11.80 ± 0.21 Hz at annuli -3, -2, and -1. Lateral thread stimulation of annuli -2 and -1 evoked 7 spikes
at 28.27 ± 0.46 Hz and 12 spikes at 29.29 ± 0.21 Hz (Figure 5.a). Ventromedial thread stimulation at
annuli -4, -3, and -2 resulted in 3 spikes at 29.41 ± 0.21 Hz, 6 spikes at 19.65 ± 0.31 Hz, and 10 spikes at
27.46 ± 0.32 Hz.
Probe Stimulation
To determine the response of neuron 3 to a harder stimulus, we stimulated the skin with a
dissecting probe. Stimulation of the dorsomedial column at annuli -5, -4, -3, -2, -1, and 0 resulted in 5
spikes at 9.01 ± 0.21 Hz, 2 spikes at 19.23 Hz, 1 spike, 1 spike, 2 spikes at 2.37 Hz, and 2 spikes at 20.20
Hz. Lateral column stimulation on the same annuli resulted in 7 spikes at 34.83 ± 0.42 Hz, 9 spikes at
35.96 ± 0.45 Hz, 7 spikes at 29.34 ± 0.62 Hz, 13 spikes at 45.41 ± 0.32 Hz, 8 spikes at 33.45 ± 0.41 Hz,
and 3 spikes at 8.75 ± 0.72 Hz (Figure 5.b). Ventromedial stimulation of annuli -5, -4, -3, -2 and -1
elicited 7 spikes at 27.78 ± 0.42 Hz, 9 spikes at 31.34 ± 0.32 Hz, 10 spikes at 31.41 ± 0.31 Hz, 13 spikes
at 39.80 ± 0.31 Hz, and 6 spikes at 17.15 ± 0.41 Hz.

Discussion
We successfully recorded from three separate mechanosensory neurons in a single leech
ganglion. From the stimulation response data, we generated a representative receptive field diagram
including thread and probe stimulation frequency responses for each of the three neurons (Figure 6). In
observation of the diagram, it is clear that our initial hypothesis that the receptive field of the neurons
would be strongest at the 0-annulus with response decreasing symmetrically to stimuli in the positive and
negative directions was generally incorrect. While neuron 2 had the strongest frequency response at the 0-
annulus, the other neurons had the strongest response at annuli other than 0. In addition, all of the neurons
showed either rostral or caudal receptive fields as opposed to symmetric about their ganglion.
While we were unable to collect full receptive field data for neuron 1, the generally local
response and the fact that the neuron responded to both the thread and the probe suggests that it is
encoding local pressure changes as would be suggested by the neuron’s location in the ganglion. The fact
RECORDINGS OF LEECH MECHANOSENSORY NEURONS 7

that neuron 2 only responded to the probe and had a relatively large receptive field suggests that it was a
nociception neuron. The tonic sustained stimulus response further suggests that neuron 2 is a rapidly
adapting N-neuron (Figure 4). Finally, neuron 3 had a relatively large receptive field and responded to
both the thread and probe which is consistent with that neuron being a touch-sensitive neuron.
Observing the size of the spatial receptive fields, we can qualitatively confirm our hypothesis that
pressure neurons would have a smaller spatial receptive field than touch and nociception neurons. This is
due to the fact that touch and nociception neurons branch throughout the body wall whereas pressure
neurons have a morphology such to detect local pressure changes (De-Miguel, 1997).
The spatial, temporal, and qualitative receptive field of a neuron determines its ability to encode
and differentiate between different types of stimuli. As observed from our experiments, some of the
neurons had overlapping spatial receptive fields (neuron 1, 2), however their multi-dimensional receptive
fields were very different allowing them to encode different types of stimuli (pressure and pain). The
ability to differentiate between harmful and benign stimuli as well as the ability to encode a stimulus
intensity is crucial for an organism’s survival. If an organism wasn’t able to differentiate between a light
touch and a threatening jab, the organism would wither expend valuable resources escaping every
stimulus or would not elicit an escape response when a harmful stimulus is present. An interesting future
experiment would be to record from motor neurons during same stimuli presented during these
experiments to determine how the ability to differentiate sensory stimuli causes variability in motor
response.
RECORDINGS OF LEECH MECHANOSENSORY NEURONS 8

References
Bosma, M., Perkel, D., Kennedy, M., Canfield, J., Hass, C., Sisneros, J. (2017). Introduction to Systems
and Behavioral Neurobiology.
Carlton, T., & McVean, A. (1995). The role of touch, pressure and nociceptive mechanoreceptors of the
leech in unrestrained behaviour. Journal of Comparative Physiology A: Neuroethology, Sensory,
Neural, and Behavioral Physiology, 177(6), 781-791.
De-Miguel, F. F. (1997). Outgrowth patterns and directed growth of identified neurons induced by native
substrates in culture. The Journal of comparative neurology, 380, 1-15
Muller, K. J., Nicholls, J. G., & Stent, G. S. (1981). Neurobiology of the Leech. Cold Spring Harbor
Laboratory.
Titlow, J., Majeed, Z. R., Nicholls, J. G., & Cooper, R. L. (2013). Intracellular Recording, Sensory Field
Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis. Journal of
visualized experiments: JoVE, (81).
RECORDINGS OF LEECH MECHANOSENSORY NEURONS 9

Figure 1: Location and Type of Recorded Neurons – a) First recorded neuron in blue, second recorded
neuron in red, and third recorded neuron in green. b) Ganglion map from Muller et al., 1981 showing
neuron 1 is a P cell, neuron 2 is an N cell, and neuron 3 is a T cell.
RECORDINGS OF LEECH MECHANOSENSORY NEURONS 10

Figure 2: Neuron 1 Responses to Stimulation – a) Neuron 1 had a spike amplitude of 52.6 ± 0.4 mV, a
half width of 4.0 ± 0.1 ms, and spontaneous firing rate of 0.54 ± 0.32 Hz. Thread stimulation in the
dorsomedial and lateral columns didn’t elicit a change from spontaneous firing rate. b) Thread
stimulation in the ventromedial column elicited a response of 5 spikes at 22.41 ± 2.3 Hz and 2 spikes at
11.11 Hz at annulus 0 and 1, respectively. c) Probe stimulation of ventromedial annuli -4, -3, and -2
elicited 10 spikes at 26.09 ± 2.60 Hz, 13 spikes at 25.81 ± 1.90 Hz, and 22 spikes at 24.10 ± 2.1 Hz,
respectively.
RECORDINGS OF LEECH MECHANOSENSORY NEURONS 11

Figure 3: Neuron 2 Responses to Stimulation – a) Neuron 2 had a spike amplitude of 70.9 ± 0.2 mV, a
half width of 3.4 ± 0.1 ms, and spontaneous firing rate of 0.18 ± 0.08 Hz. Thread stimulation didn’t cause
the neuron to change from spontaneous firing rate. b) Dorsomedial probe stimulation at annuli -3, -2, 0,
1, 2, and 3 elicited 4 spikes at 16.30 ± 0.50 Hz, 1 spike, 5 spikes at 18.69 ± 0.42 Hz, 5 spikes at 11.87 ±
0.61 Hz, 3 spikes at 14.36 ± 0.23 Hz, and 2 spikes at 1.04 Hz. Spontaneous activity increased to 1.70 ±
0.51 Hz. Probe stimulation of the lateral wall at annuli 0, 1, 2, and 3 elicited 7 spikes at 18.05 ± 0.30 Hz,
9 spikes at 17.89 ± 0.31 Hz, 5 spikes at 13.29 ± 0.45 Hz, and 3 spikes at 18.26 ± 0.42 Hz, respectively. c)
Contralateral stimulation failed to evoke a response.
RECORDINGS OF LEECH MECHANOSENSORY NEURONS 12

Figure 4: Neuron 2 Response to Prolonged Stimulation – a) Prolonged stimulation at the lateral 0-

annulus increased the spike frequency from the spontaneous rate to 6.0 ± 0.1 Hz. The response frequency
decreased throughout the sustained stimulus. b) Rate meter showing counts/bin in 2 second bins before,
during, and after the stimulus.
RECORDINGS OF LEECH MECHANOSENSORY NEURONS 13

Figure 5: Neuron 3 response to Stimulation – a) Neuron 3 had a spike amplitude of 33.6 ± 0.6 mV, a
half width of 3.3 ± 0.1 ms, and spontaneous firing rate of 0.33 ± 0.05 Hz. Lateral thread stimulation of
annuli -2 and -1 evoked 7 spikes at 28.27 ± 0.46 Hz and 12 spikes at 29.29 ± 0.21 Hz. b) Ventromedial
probe stimulation of annuli -5, -4, -3, -2 and -1 elicited 7 spikes at 27.78 ± 0.42 Hz, 9 spikes at 31.34 ±
0.32 Hz, 10 spikes at 31.41 ± 0.31 Hz, 13 spikes at 39.80 ± 0.31 Hz, and 6 spikes at 17.15 ± 0.41 Hz.
RECORDINGS OF LEECH MECHANOSENSORY NEURONS 14

Figure 6: Mapping of Neuronal Receptive Fields – All annuli that elicited a response that we were able
to record are labeled with the response frequency of that neuron. Neuron 1 is represented in blue, neuron
2 is represented in red, and neuron 3 is represented in green. Thread stimulation is represented by a
lighter shade and probe stimulation is represented by a darker shade.