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Effects of Cooling on the Activity of Neurons in the Inferior Colliculi

Auditory Group, Institute of Neuroscience, The Medical School, Newcastle upon Tyne, NE2 4HH
l.d.orton@ncl.ac.uk

Llwyd Orton, Paul Poon and Adrian Rees
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Brain cooling allows rapidly reversible inactivation of neural activity. It has been used for fifty years to investigate the nature of audition since Misrahy et al. (1960) placed ice on the surface of the brain and recorded the effects on cortical evoked potentials [1]. More recently Lomber et al. (1999) developed the ‘cryoloop’, providing a more precise method of cooling neural tissue [2]. The inferior colliculi (IC) are two cupola shaped structures protruding dorsally from the tectal midbrain. They receive auditory information from numerous brain stem nuclei along with descending thalamocortical connections. The colliculi are connected by the commissure of the inferior colliculus (CoIC). The fibres within the CoIC are tonotopically organised A allowing neurons within one frequency band lamina to innervate the corresponding lamina contralaterally.

Introduction

Single Unit Results cont.
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Little is known of the functional significance the CoIC. We aim to use tissue cooling to investigate the mechanisms by which one IC exerts control over the other via the CoIC. Here we address how direct cooling influences activity in the IC and modulates responses in the opposite IC. By combining cooling with commissurotomy we test the hypothesis that cooling effects in the contralateral IC are mediated through the CoIC.

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(A) Horizontal sections of the IC’s stained to show the myelinated fibres of the CoIC [3]. (B) High magnification of the CoIC stained in A.

When cooled, one group of neurons showed an exponential decrease in firing rate (e.g. A). A second group were characterised by an initial increase in firing rate during the first 10°C of cooling followed by a decrease (eg. B). (C) Data from 38 neurons recorded in the directly cooled IC. The firing rate on cooling to <10°C was significantly different from the control and recovered conditions (two tailed t-test, P<0.001). (D) In 20/38 units firing rate increased during the first 10°C of cooling. This increase in firing was significantly different from the responses of the remaining units (P<0.001).

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Single units in IC modulated by cooling the contralateral IC. (A) shows change in firing rate for two units in the recorded IC during cooling with their frequency response areas (B-D) and (E-G) respectively. The response of one cell (blue) initially decreases and then increases as temperature is reduced below 30°C. The other unit (red) shows the reverse change.

We investigated the effects of cooling using single unit and local field potential recordings in the directly cooled IC and its contralateral counterpart. Adult guinea pigs (400-900g) were anaesthetised with urethane and Hypnorm. The animal was placed in a stereotaxic frame with the ear bars replaced by hollow specula into which headphones were inserted allowing sound stimuli to be delivered to the animal. A craniotomy was performed & the left occipital lobe aspirated to expose the left IC. Cooling was achieved by pumping cold ethanol through a stainless steel loop, placed in contact with the lateral edge of the left IC (Fig, 2). A thermocouple allowed continuous monitoring of the probe temperature. The probe temperature was never allowed to fall below 1°C to prevent trauma to the tissue. Single units were recorded in the central nucleus of the IC (CNIC) with a tungsten micro electrode in response to 100ms-duration tone bursts.

Methods

• Previous studies have applied large temperature changes but have not investigated smaller cooling steps. • While the overall effect of cooling to ~10°C was always a marked reduction in firing rate (mean 78%, Fig.4A&C), a novel finding was that in over half of units, more moderate cooling in temperature (5-10°C below physiological temperature) produced an increase in firing rate (Fig, 4B&D). These increases varied from 1.07 to 1.86 of pre-cooled values. • Such responses may reflect selective changes in synaptic input, or direct effects of cooling on membrane conductances

E ect of IC cooling on responses in the contralateral IC:
• Single unit recordings were made from 11 units in the IC contralateral to the cooled IC. • The firing rates of all units were modulated by cooling the opposite IC. • Seven units showed an increase in responsivity, the other four decreased their firing. • Seven of these eleven showed a two phase response (Fig. 5) evident in both their PSTH spike count and frequency response area.

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Evoked Potential Results
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Figure 6
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Each unit’s frequency-response area and PSTH were recorded throughout a step wise cooling.

(A) Coronal section through the preparation showing the location of the cryoloop and ipsilateral micro electrode placement. (B) Schematic of the cooling system [adapted from 4]. Ethanol was drawn from a reservoir via a peristaltic pump. It is cooled by ethanol at ca. -80°C before passing through the cryoloop.

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Evoked potentials recorded over the right IC under three stimulus conditions: (A) Left ear only; (B) Right ear only; (C) Binaural (Fig. 6). Dashed line indicates onset of IC response estimated from single unit latency. Cooling the left IC to 29°C, led to larger responses to left and binaural stimulation, and a reduced response to right ear stimulation. With cooling to 5°C, responses were reduced regardless of stimulation paradigm. (D) Area under the curve for all three stimulation paradigms. The response to left ear stimulation increased with cooling to 29°C and decreased at 5°C, whereas there was little change to right ear stimulation. (E) The first deflection after 6ms likely indicates activity in the IC. An index of change shows that this peak follows the trend seen in (D). (F) Latencies of the same peak during cooling and recovery. Cooling to 5°C increased the latency of response.

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Evoked potential recordings were made in response to a 1ms, 10kHz tone pip, either with a silver ball electrode placed on the dorsal surface of the un-cooled IC or with a micro electrode placed in the right IC .

Figure 7
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Single Unit Results
Direct cooling e ects:
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• 38 units were recorded in the directly cooled IC. • Large reductions in temperature always decreased neuronal spiking activity. • Figure 3 shows the response of a single neuron to 3°C step changes in temperature. • The unit maintains the same frequency tuning but spike activity decreases during cooling (Fig. 3A & PSTHs B-D). • On rewarming a rapid recovery in firing rate is coupled with increased spontaneous activity evident in the frequency response area plots.

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Evoked potentials recorded from an electrode within the IC while the opposite IC was cooled to 5°C. (A) Left ear only; (B) Right ear only; (C) Index of peak change To determine whether the effects seen in Figure 6 were mediated by the CoIC, we recorded gross activity from the IC before and following transection of the CoIC during cooling. Three peaks were produced to left ear stimulation (A). The first likely indicates incoming brain stem activity. This peak increased slightly when cooled with little further change after the commissurotomy. The second peak reduced by > 50% during cooling (red). This reduction was almost abolished post commissurotomy. (B) With right ear stimulation cooling resulted in a reduction in peak magnitude (red) with a further reduction following commissurotomy (blue). (C) Summary of effects shown in A & B. (D & E Left) Schematic of the paradigm implemented in A & B. (D Middle) reducing activity in the left IC by cooling reduced the potential observed (red trace in A). (D Right) cutting commissural fibres abolished this inhibitory effect from reaching the electrode. (E Middle) The indirect inhibition of local potentials within the right IC due to cooling the left IC. (E Right) Commissurotomy completely removes the CoIC input. The remaining response is due to ipsilateral input.

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(A) Neuronal firing rate during cooling and rewarming. Insets are frequency response areas at five stages of cooling. The responsiveness of the cell decreased during cooling and increased once cooling was stopped. (B) PSTH of the same unit pre-cooling with an on-sustained pattern. (C) Cooling suppresses neural output to 15% of pre-cooled level. (D) The cell’s firing recovers to within 5% of the pre-cooled spike count when cooling is stopped.

• Evoked potential recordings identified the effects of cooling on population activity. • Because the predominant source of excitation to each IC is the contralateral ear, we could dissociate the effects of the CoIC by comparing responses to monaural and binaural stimuli. • The evoked potential in the right IC to left ear stimulation increased when the left IC was cooled to 29°C and decreased when cooled to 5°C. Smaller changes were seen with right ear stimulation. • These findings suggest that activation of the CoIC is dependent on the pattern of ear stimulation (Fig. 7D & E).
1) Direct cooling of the IC reduces neuronal activity when probe temperature falls below 30°C. More moderate cooling results in increased activity in about half of units. 2) Activity changes in the IC contralateral to the cooled IC are consistent with the CoIC mediating both facilitatory and inhibitory effects. 3) Both evoked potentials and single units recorded in the IC opposite to the cryoloop undergo temperature dependent modulation suggesting the, CoIC influences both individual neurons and population network activity. 4) Gross potential recording combined with cooling and commissurotomy suggests that the commissural influence on the IC is dependent on the ear stimulated.
1) Misrahy GA, Spradley JF, Reran AV & Garwood VP. (1961). Acoustic cerebellar pathways in cats. Journal of Neurophysiology 24, 159-66. 2) Lomber SG, Payne BR & Horel JA. (1999). The cryoloop: an adaptable reversible cooling deactivation method for behavioral or electrophysiological assessment of neural function. Journal of Neuroscience Methods 86, 179-194. 3) Saldaña E, Merchan MA. (1992). Intrinsic and commissural connections of the rat inferior colliculus. The Journal of Comparative Neurology 319, 417-437. 4) Salsbury KG, Horel JA. (1983). A cryogenic implant for producing reversible functional brain lesions. Behaviour Research Methods & Instrumentation 15, 433–6.

Conclusions

Acknowledgements
Supported by a studentship from Newcastle University

References