February 2003
Volume 44, Issue 2
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Physiology and Pharmacology  |   February 2003
Activation of Scleral Cold Thermoreceptors by Temperature and Blood Flow Changes
Author Affiliations
  • Juana Gallar
    From the Institute of Neurosciences, Miguel Hernández University Superior Council for Scientific Research, San Juan de Alicante, Spain.
  • M. Carmen Acosta
    From the Institute of Neurosciences, Miguel Hernández University Superior Council for Scientific Research, San Juan de Alicante, Spain.
  • Carlos Belmonte
    From the Institute of Neurosciences, Miguel Hernández University Superior Council for Scientific Research, San Juan de Alicante, Spain.
Investigative Ophthalmology & Visual Science February 2003, Vol.44, 697-705. doi:10.1167/iovs.02-0226
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      Juana Gallar, M. Carmen Acosta, Carlos Belmonte; Activation of Scleral Cold Thermoreceptors by Temperature and Blood Flow Changes. Invest. Ophthalmol. Vis. Sci. 2003;44(2):697-705. doi: 10.1167/iovs.02-0226.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To study the response of scleral cold receptors located in areas of the eye unexposed to temperature and blood flow changes.

methods. In anesthetized cats, the neural activity was recorded from single, cold-sensory fibers of the ciliary nerves innervating the sclera and limbus. Controlled temperature changes of the receptive field were performed with a contact thermode. Ocular blood flow reductions were obtained by occluding the ipsilateral common carotid artery for 30 to 60 seconds with a compressor placed around the artery. Local blood flow was measured with a laser Doppler flowmeter. Temperature was measured with a microprobe introduced in the subscleral space. Ocular sympathetic stimulation was performed with a pair of silver electrodes placed on the preganglionic cervical sympathetic trunk. To induce local hypoxia, N2 was applied on the scleral surface with a specially designed chamber. For systemic hypoxia the breathing air was replaced with a gas mixture containing 10% O2 in N2.

results. Sensory nerve fibers identified as cold receptors exhibited ongoing nerve activity in bursts at 35°C and responded to cooling pulses applied to their receptive fields with an increase in the impulse discharge that reached a peak and decayed gradually to a lower level. When temperature was reduced from 35°C to 34°C, frequency increased monotonically with decreasing temperature of the sclera. Between 35°C and 30°C, peak and mean frequencies were roughly proportional to temperature of the sclera. The characteristics of burst discharges also depended on scleral temperature. Electrical stimulation of the cervical sympathetic trunk induced a decrease in blood flow and temperature and evoked an increase in the firing frequency of cold-sensory fibers that was proportional to the frequency of stimulating pulses. Carotid occlusion also elicited an increase of the discharge of cold thermoreceptor fibers that occurred in parallel with a decrease in blood flow and temperature in the receptive field area. Local or systemic hypoxia did not modify appreciably the spontaneous firing frequency of scleral cold-sensory fibers.

conclusions. Scleral and episcleral cold-sensory fibers encoded as a change in their impulse frequency and firing pattern temperature reductions of less than 1°C in scleral tissues. Activation of scleral and episcleral cold-sensory fibers by sympathetic vasoconstriction and acute arterial pressure reductions appear to be secondary to the temperature decrease that accompanies the reduction in ocular blood flow caused by these maneuvers. Scleral thermosensory fibers are located in ocular territories not directly exposed to external temperature changes. Thus, the sensory information on local blood flow variations provided by these receptors may be involved in a reflex regulation of choroidal blood flow that functions to maintain a constant temperature and blood supply to the retina.

In the eye of the cat, several functional types of sensory receptors have been distinguished according to their response to non-noxious and noxious mechanical, chemical, and thermal stimuli. 1 2 3 4 Thermal receptors activated by cold are relatively abundant in areas of the eye that are exposed to external temperature changes, such as the cornea, the limbus, and a portion of the bulbar conjunctiva. 2 5 They appear to be primarily involved in detecting external temperature variations, including those associated with tear evaporation. 6 7  
In addition, cold-sensory fibers with receptive fields located in the intraorbital sclera or the iris have been incidentally described. 2 8 9 The functional significance of thermal receptors supplying ocular structures that are largely protected from environmental temperature changes 10 remains obscure. It has been hypothesized that these cold receptors could be involved in the detection of local blood flow changes. 8 In the present work, the characteristics of nerve impulse discharges in scleral cold thermoreceptors and their modification by local temperature blood flow changes were explored. Preliminary results have been reported elsewhere. 11  
Materials and Methods
Experiments were performed in 14 adult cats of either sex anesthetized with pentobarbital sodium (40 mg/kg, intraperitoneally; Nembutal; Abbott Laboratories, Abbott, IL). They were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Animals breathed spontaneously through a tracheal cannula and were kept in an areflexic state during the experiment, by intravenous infusion of dilute anesthetic (3 mg/kg per hour) with a perfusion pump. End-tidal CO2 (3%–5%) was continuously monitored. Rectal temperature (37–38°C) was maintained within the physiological range with a servoregulated heating device. At the end of the experiment, cats were killed with an overdose of anesthetic. 
The technique for extracellular recording of single ocular afferent fiber activity has been described previously. 1 In brief, the ciliary nerves were dissected in the back of the eye, and fine nerve filaments were subdissected and placed on Ag-AgCl electrodes. Impulse activity of single units was recorded and amplified with conventional electrophysiological equipment. Scleral fibers sensitive to temperature were initially identified by their spontaneous firing, usually in bursts that increased in frequency and duration when a brush wetted in ice-cold saline was slid over the scleral surface. Receptive fields of cold units were mapped with the tip of an ice-cooled metal bar (0.5 mm diameter). Cold-sensitive fibers were unresponsive to mechanical stimulation with von Frey hairs (0.1–1 N). 
Conduction velocity was calculated from the latency of suprathreshold electric shocks (0.1–0.5 ms, 0.5–3 mA) applied to the receptive field with a bipolar Ag-AgCl electrode, and by measuring the conduction distance between the stimulating and the recording points with a 6.0-gauge thread placed along the presumed trajectory of the nerve. 
Controlled temperature changes of the ocular surface were performed with a contact thermode (tip size: 4.5 mm2), made of a water-cooled Peltier cell regulated by feedback circuitry that maintained the temperature of the probe within ±0.05°C of the desired value. 2 At room temperature (24°C), the temperature of the surgically exposed sclera was 31.7 ± 0.2°C (n = 12), 2°C lower than scleral temperature measured before surgical removal of lids, orbital wall, and extraocular muscles to expose the ciliary nerves (33.7 ± 0.1°C, n = 12). Temperature stimuli consisted of cooling pulses of 15-second duration and increasing magnitude (temperature decreases of −2°C up to −30°C from an adapting temperature of 35°C) applied at 2-minute intervals. The rate of temperature decrease at the tip of the probe was 0.7°C/sec and 0.27°C/sec when measured inside the sclera, just below the probe. 
Real-time measurements of ocular tissue blood flow were performed with a laser Doppler flowmeter (floLAB; Moor Instruments, Ltd., Axminster, UK) provided with an angled probe (model MP5a, center fiber separation of 0.5 mm) that was gently placed onto the ocular surface by means of a micromanipulator, covering the receptive field of the recorded unit. Flux signals were analyzed with the accompanying software (moorLAB for Windows, version 1.1; Moor Instruments, Ltd.). Blood flow was measured in arbitrary units and the changes expressed as a percentage variation from the baseline flow. 
Measurements of ocular temperature changes during carotid occlusion and sympathetic stimulation were performed with a 0.003-in. (76 μm) diameter thermocouple microprobe (Type IT-23, Physitemp Instruments, Inc., Clifton, NJ). Temperature changes associated with blood flow changes were measured with the probe introduced into the subscleral space through a small hole performed in the exposed sclera. In a set of experiments, temperature changes were measured in the sclera protected by lids and orbit. 
Ocular blood flow reductions were obtained by occluding the ipsilateral common carotid artery for 30 to 60 seconds with a compressor made of a 1-mm diameter silk filament covered by a soft silicone tube (outside diameter: 1.25 mm) placed around the artery with both ends passed through a larger polyethylene tube (3 mm inside diameter). Pulling the ends of the silk filament occluded the artery without stretching it. 12  
Ocular sympathetic stimulation was performed with a bipolar silver electrode placed around the preganglionic cervical sympathetic trunk 13 and applying 10-V, 0.5-ms electrical pulses at variable frequency (5–25 Hz) during 20 to 30 seconds. The efficacy of the stimulation was verified by the mydriasis evoked by the stimulus. 
Local hypoxia was produced with an atmosphere of pure N2 created by placing a 16-mm diameter polypropylene chamber filled with 100% N2, attached to the scleral surface with silicone grease, on the area of the sclera that contained the receptive field. N2 (100%) was made to flow continuously into the chamber for 1- to 2-minute periods. Systemic hypoxia was obtained by replacing the breathing air with a gas mixture containing 10% O2 in N2 for 30- to 60-second periods. 
Neural discharges, temperature of the stimulating contact thermode, blood flow, and temperature changes were simultaneously recorded on a magnetic tape and analyzed off-line with an analog-to-digital converter (1401Plus; CED, Ltd., Cambridge, UK) and accompanying software (Spike2 for Windows version 3.13; CED, Ltd.). Mean discharge rate (impulses per second [imp/sec]) and instantaneous firing frequency were measured to evaluate the impulse response of scleral cold units to the different experimental maneuvers (thermal stimulation, carotid artery occlusion, electrical stimulation of the ocular sympathetic nerves and hypoxia). During thermal stimulation, the responses to the ramp of the cooling pulse (dynamic response) and to the steady, final temperature (static response) were analyzed separately. Data are expressed as the mean ± SEM. Paired t-test and one-way ANOVA were used for statistical comparison of data, as indicated. 
Experimental Protocol
When a nerve filament containing multiunit activity that varied in response to the stimulation of the sclera was identified, single units displaying ongoing activity were sought by subsequent splitting of the nerve strand. Localization of the receptive field of a single cold-sensory unit was made with a wet, fine brush applied to the scleral surface. The receptive field borders were then mapped with an ice-cooled metal bar and the conduction velocity measured. The tip of the thermode probe at 35°C was placed on the receptive field and, after an adaptation time of 5 minutes, the ongoing discharge at this temperature was recorded continuously. From the adapting temperature of 35°C, 15-second cooling pulses of increasing magnitude (−2°C up to −30°C, in 2°C steps) were sequentially applied, with 2-minute intervals between pulses. After characterization of the response to cold, carotid artery occlusion, electrical stimulation of the sympathetic trunk, and local and systemic hypoxia were made with at least 5-minute intervals between maneuvers. In some units, the stimulation protocol was repeated after a 15-minute resting period. 
Results
General Properties of Cold-Sensory Fibers
Nerve impulse activity in 76 scleral cold-sensitive units was recorded. The conduction velocity, measured in 49 units ranged from 0.6 to 6.5 m/sec (mean: 2.5 ± 0.2 m/sec). Based on their conduction velocity, units were classified as Aδ (conduction velocity ≥ 1.5 m/sec, n = 36) or C fibers (conduction velocity < 1.5 m/sec, n = 13). 
The receptive fields of cold receptor fibers were usually round and small, with a mean diameter of 1.8 ± 0.1 mm (n = 47). They were located in the anterior episclera (n = 52), the limbus (n = 17), and the posterior sclera (n = 7). In 41 units, the receptive field was examined under the surgical microscope. In all instances, it was located very near or over the trajectory of a blood vessel. Figure 1A offers an example of the various locations on the eye surface of the receptive field of cold-sensory fibers in the sclera, the limbus, and the perilimbal episclera. Most of them are normally covered by the lids in the awake animal (Fig. 1B)
Thermal Stimulation
Almost all cold units included in this study (74/76) exhibited spontaneous impulse activity at 32°C (mean frequency measured in 36 units: 8.4 ± 0.8 imp/sec, range, 0.5 to 20.6 imp/sec). At this temperature, 75% of the spontaneously active units fired rhythmically in bursts composed by 2 to 11 impulses separated by intervals of total silence (Fig. 2A) . When an ice-cooled bar was placed on the receptive area or a drop of saline at 25°C was applied, a continuous discharge of impulses of higher frequency was transiently evoked, followed by a more irregular firing pattern or more often, by a brief period of silence. 
All units increased their firing rate in response to a controlled reduction of the temperature of their receptive field from an adapting temperature of 35°C. Figure 2B shows the typical impulse discharge of a cold unit in response to a cooling pulse of −14°C below the adapting temperature. The firing frequency increased sharply during the initial part of the temperature step (dynamic component of the response) and then stabilized at a lower steady state frequency level (static component of the response). Figure 3 presents an example of the values of instantaneous frequency evoked by three cooling pulses of increasing magnitude, to illustrate the rapid change in impulse firing, the shift to a prominent bursting pattern, and the high peak frequency attained during the dynamic component of the response. When temperature reached a final level, the impulse discharge became transiently continuous and then resumed rhythmic firing at a new steady state mean frequency. Figure 3 also presents the histograms of impulse interval distribution at different temperatures that evidence the increase in peak frequency value with progressively larger cooling pulses. 
Figure 4A shows the stimulus-response curves of the static and dynamic responses of 11 units, evoked by 15-second cooling pulses of increasing magnitude (−2°C up to −30°C) from an adapting temperature of 35°C. Scleral cold fibers increased their firing rate significantly during thermal stimulation for all the explored temperatures (one-way ANOVA). Mean frequencies (impulses per second) were higher during the dynamic part of the discharge. Also, temperature changes were best encoded between 35°C and 30°C, whereas at temperatures below 10°C, impulse firing was markedly reduced. 
To measure the encoding capacity of temperature decreases by scleral cold fibers, we calculated the number of impulses per second per degree Celsius (°C) of temperature reduction during cooling pulses applied at a constant rate of 0.27°C/second, changing the scleral temperature from 35°C to 34°C. As shown in Figure 4B , over a range of −1°C the discharge frequency evoked by the temperature decrease was linearly related to the temperature’s decline (1.7 ± 0.1 imp/sec per 0.1°C, n = 11). The organization of nerve impulse bursts in cutaneous cold thermal receptors has been claimed to provide a measure of the steady temperatures at the skin surface. 14 Thus, in four scleral cold fibers, the number of impulses per burst, the burst duration, and the interburst intervals were measured at various steady temperatures. As shown in Figure 5 the three parameters were related to steady temperatures between 35°C and 27°C. 
Sympathetic Stimulation
The effect on nerve impulse frequency of an electrical stimulation of the cervical sympathetic trunk was explored in four units, by using increasing stimulation frequencies (5, 10, 15, 20, and 25 Hz; Fig. 6A ). Electrical stimulation at different frequencies produced a transient increase in ocular blood flow and subscleral temperature, accompanied by a silent period in cold receptor activity (Fig. 7) that was followed by a significant reduction in blood flow (−72% ± 21%, n = 6) and temperature (−0.47 ± 0.08°C, n = 3; Figs. 6B 6C ) that persisted up to 200 seconds after the end of the stimulation (Figs. 6B 6C) . Blood flow and temperature decreases were roughly proportional to the frequency of stimulation (r 2 = 0.52 and 0.47 for blood flow and temperature, respectively). In all cases, the spontaneous firing frequency increased significantly (mean for the four units: 9.7 ± 1.5 imp/sec basal frequency to 19.3 ± 2.4 imp/sec during sympathetic stimulation, n = 4, P < 0.05, one-way ANOVA on ranks; Fig. 6A ) and in a manner proportional to the frequency of stimulation (r 2 = 0.74). In the sclera covered by the lids, electrical stimulation of the sympathetic nerve also induced a significant temperature reduction (−0.13 ± 0.04°C, P < 0.05, paired t-test, n = 7) that showed similar time course and lower magnitude than in the exposed sclera (Fig. 8A)
Carotid Artery Occlusion
Occlusion of the carotid artery for 30 to 60 seconds increased significantly the firing frequency in 25 of 27 units (from 9.7 ± 1.4 to 14.3 ± 1.6 imp/sec, n = 27, P < 0.05, one-way ANOVA on ranks; Fig. 9A ). This change was not significant when receptive field temperature was held constant at 33°C with the thermode during the carotid occlusion (13.2 ± 5.3 and 15.3 ± 6.2 imp/sec before and during occlusion, respectively, n = 5). 
Local blood flow decreased immediately after the onset of the occlusion (Fig. 10B) to almost half of the control value (190.2 ± 31.9 to 101.1 ± 13.7 AU, n = 31, P < 0.05 one-way ANOVA on ranks; Fig. 9B ) and recovered slowly after interruption of the occlusion (Fig. 10B) . Changes in firing frequency of scleral cold fibers roughly followed those of the blood flow traces (Fig. 10) . Similarly, at the end of the carotid occlusion the basal impulse frequency recovered with the return of blood flow to control levels (Figs. 9A 9B) . Carotid artery occlusion also produced a significant and immediate decrease in temperature (−0.33 ± 0.03°C, n = 3, P < 0.01, paired t-test) that outlasted the duration of the occlusion (Figs. 9C 10B) . A smaller but significant temperature decrease (−0.20 ± 0.04°C, n = 5, P < 0.05, paired t-test) was also observed during carotid occlusion in the sclera covered by the lids (Fig. 8B , open circles). 
Hypoxia
In seven cold units, exposure of the receptive field to an atmosphere of 100% N2 for 1 to 2 minutes did not modify appreciably the impulse activity (10.4 ± 3.4 and 9.4 ± 2.5 imp/sec before and during local hypoxia, respectively). Systemic hypoxia, explored in three additional cold fibers also failed to change the ongoing firing frequency (9.3 ± 4.9 and 9.3 ± 5.1 imp/sec before and during systemic hypoxia, respectively). 
Discussion
Our results show that cold thermoreceptors sensitive to small temperature changes are distributed throughout the entire ocular surface, most frequently in the highly vascularized perilimbal episclera, but also in the posterior segment of the eye. Nerve fibers activated by cold had spotlike receptive fields and responded to temperature reductions with a nerve impulse discharge that started as soon as the local temperature began to decline, and increased monotonically in firing frequency in parallel with the decrease in temperature. 
The functional properties of cold nerve endings placed in the outer coats of the eye were closely similar to those found in cold afferent fibers innervating other trigeminal territories such as the tongue or the skin of the face. These are Aδ or C fibers that respond to local declines in the temperature in the tissues where nerve terminals are located 15 and exhibit a discharge in bursts, showing static and dynamic sensitivity curves with maxima around 30°C. 16 17 18 19 20 21 . Cutaneous and lingual thermoreceptors are most sensitive to thermal transients over a limited temperature range that lies between 30°C and 26°C, 22 but also encoded steady temperatures through changes in their bursting pattern. 14 Stimulation of sensory or autonomic nerves of the skin modifies cold thermal receptor activity, a change attributed to variations in cutaneous blood flow. 23  
Similarly, in the outer coats of the eye, cold receptors were primarily Aδ fibers that fired in bursts, produced high instantaneous frequency peaks, and encoded progressive cooling, as an accelerating discharge of impulses in a range similar to trigeminal cold receptors of other territories. The mean impulse discharge of scleral cold-sensory fibers signaled poorly steady temperatures although the organization of their bursting pattern was different for the different temperatures, as is the case for cold receptors of other tissues. Many of these functional properties have been also observed in cold-sensitive fibers of the feline cornea, 2 with minor differences in the firing pattern attributable to the particular structure of the corneal tissue. Therefore, it can be concluded that ocular cold receptors do not differ functionally from thermal receptors of other territories of the body. 
Scleral cold fibers were also activated by reductions in ocular blood flow obtained either directly through a carotid artery occlusion or secondary to vasoconstriction induced by electrical stimulation of the cervical sympathetic trunk. The effects of reductions in local blood flow on the firing frequency were prominent. There, as in the skin 23 excitation of cold-sensory endings can be explained by a direct effect of temperature that is expected to decline secondary to the decrease in blood flow. 24 25 The parallelism observed between blood flow reductions and local temperature supports this interpretation. The transient silent period in the impulse discharge that occurred at the onset of sympathetic stimulation, coincident with a transient increase in blood flow and temperature, also speaks in favor of a direct effect of temperature on cold nerve endings. In the present experiments, blood flow measurements presumably corresponded to the sclera and the underlying choroidal blood vessels. In a recent morphologic study, 26 nerve terminals of functionally identified scleral cold-sensory fibers were localized within the dense collagenous tissue of the sclera. Nerve endings were not closely associated with small scleral blood vessels, which are relatively scarce. Although in our study, receptive fields were often located near large blood vessels running on the scleral surface the main temperature variations around cold nerve endings caused by ocular blood flow reductions possibly originate in the underlying, richly vascularized choroid. 
Other possible mechanisms, apart from temperature reductions, to explain cold receptor activation by reduced flow, appear to be less likely. It is doubtful, for instance, that the increased firing of scleral cold receptor fibers consecutive to carotid artery occlusion or sympathetic activation partly resulted from a mechanical stimulation of cold nerve terminals produced by changes in blood filling of nearby vessels. 27 Cold receptor units have, at best, low mechanical sensitivity. 2 17 Moreover, in the skin of the monkey and cat the bursting pattern of cold receptor fibers, which is independent of the heart cycle, was not affected by the rhythmic oscillations of arterial pressure. 18  
Also, tissue hypoxia can presumably be ruled out as the origin of the response of cold fibers to decreased blood flow. It has been proposed that cold receptor activation by temperature reductions is due in part to a blockade of the electrogenic Na+,K+ pump that leads to nerve terminal depolarization and increased firing. 28 29 30 In accordance with this metabolic dependence, hypoxia secondary to reduced blood flow could lead indirectly to activation of cold receptors. However, no firing of scleral cold receptor activity was evoked by short-term local and systemic hypoxia in the present work. Thus, moderate hypoxia accompanying carotid artery occlusion or sympathetic stimulation 25 27 28 31 does not seem to be sufficient to activate cold fibers through a metabolic mechanism. A small, direct effect of catecholamines on cold receptor activity has been reported in the frog’s skin. 32 Sympathetic activity was increased directly during electrical stimulation and reflexively during carotid occlusion. However, direct effect of sympathetic fibers on peripheral sensory receptors of mammals are small or nonexistent. 33 Moreover, when temperature of the scleral surface was held constant, carotid occlusion did not change the firing rate of cold fibers. Thus, a direct effect of adrenergic neurotransmitters on cold nerve endings is highly unlikely. 
The functional significance of thermal eye receptors that also respond to changes in local blood flow remains speculative. Sensory inflow from cold fibers innervating the cornea seems to contribute to conscious thermal sensations of the eye and face 6 7 8 and may also participate in blinking evoked by ocular surface evaporation. 34 The temperature around cold nerve endings located in areas of the eye surface that are minimally exposed to environmental variations, however, would be determined mainly by the local blood flow. In intact eyes, blood flow reductions evoked by sympathetic stimulation or carotid occlusion produced subscleral temperature changes of less than 1°C. Nevertheless, changes in instantaneous frequency and bursting pattern of cold fibers induced by temperature reductions of similar magnitude in the skin, evoked unambiguous sensations of cold 35 and reflex thermoregulatory responses in humans. 36 Thus, it is possible that activation of scleral cold-sensory fibers may serve to signal not only temperature reductions but also small changes in blood flow. 
Retinal cells are critically dependent on an adequate blood supply and a stable temperature. 37 38 Cold receptor fibers placed in unexposed areas of the sclera may provide the sensory input for a reflex regulation of regional temperature and blood flow, to maintain normal retinal function. 
 
Figure 1.
 
Distribution of cold thermoreceptors on the scleral surface of a feline eye. (A) Location of the receptive fields of scleral and limbal cold thermoreceptor fibers in the temporal side of the eye. (B) Front view of the feline eye illustrates that the sclera and a large part of the limbus are not exposed to external temperature changes.
Figure 1.
 
Distribution of cold thermoreceptors on the scleral surface of a feline eye. (A) Location of the receptive fields of scleral and limbal cold thermoreceptor fibers in the temporal side of the eye. (B) Front view of the feline eye illustrates that the sclera and a large part of the limbus are not exposed to external temperature changes.
Figure 2.
 
Sample recordings of impulse frequency of scleral cold receptor fibers. (A) Traces are examples of the spontaneous activity of two different cold fibers at 32°C. (B) Impulse discharge of a cold unit in response to a cooling pulse from an adapting temperature of 35°C down to 21°C (bottom trace).
Figure 2.
 
Sample recordings of impulse frequency of scleral cold receptor fibers. (A) Traces are examples of the spontaneous activity of two different cold fibers at 32°C. (B) Impulse discharge of a cold unit in response to a cooling pulse from an adapting temperature of 35°C down to 21°C (bottom trace).
Figure 3.
 
Response of a scleral cold thermoreceptor to −2°C, −4°C, and −6°C cooling pulses applied sequentially with a 2-minute interval from an adapting temperature of 35°C (top record). (A) Instantaneous frequency during a 30-second period, including the dynamic and static phases of the cooling pulse (bottom trace). (B) Interval histogram showing the distribution of the intervals between nerve impulses. Note the shift to the left with larger cooling pulses.
Figure 3.
 
Response of a scleral cold thermoreceptor to −2°C, −4°C, and −6°C cooling pulses applied sequentially with a 2-minute interval from an adapting temperature of 35°C (top record). (A) Instantaneous frequency during a 30-second period, including the dynamic and static phases of the cooling pulse (bottom trace). (B) Interval histogram showing the distribution of the intervals between nerve impulses. Note the shift to the left with larger cooling pulses.
Figure 4.
 
Scleral cold thermoreceptor response to cold stimulation. (A) Average response of 11 cold-sensory fibers during the dynamic (○) and static (•) components of the cooling pulse. Data are the mean ± SEM of the mean discharge rate. (B) Stimulus-response curve obtained from fibers shown in (A) during the dynamic change of scleral temperature between 35°C and 34°C. The firing frequency (impulses/second) was linearly related to the temperature reduction (r 2 = 0.74). Inset: relationship between stimulus temperature measured at the tip of the probe (abscissa) and temperature measured inside the sclera (ordinate) during cooling pulses (r 2 = 0.96), to illustrate the correlation between temperature reduction in the sclera and the probe tip during the dynamic component of the cooling pulse.
Figure 4.
 
Scleral cold thermoreceptor response to cold stimulation. (A) Average response of 11 cold-sensory fibers during the dynamic (○) and static (•) components of the cooling pulse. Data are the mean ± SEM of the mean discharge rate. (B) Stimulus-response curve obtained from fibers shown in (A) during the dynamic change of scleral temperature between 35°C and 34°C. The firing frequency (impulses/second) was linearly related to the temperature reduction (r 2 = 0.74). Inset: relationship between stimulus temperature measured at the tip of the probe (abscissa) and temperature measured inside the sclera (ordinate) during cooling pulses (r 2 = 0.96), to illustrate the correlation between temperature reduction in the sclera and the probe tip during the dynamic component of the cooling pulse.
Figure 5.
 
Organization of burst discharges of scleral cold thermoreceptors at different static temperatures. The number of impulses per burst (A), the burst duration (B), and the interburst interval (C) were represented versus steady temperature. Data are mean ± SEM, n = 4.
Figure 5.
 
Organization of burst discharges of scleral cold thermoreceptors at different static temperatures. The number of impulses per burst (A), the burst duration (B), and the interburst interval (C) were represented versus steady temperature. Data are mean ± SEM, n = 4.
Figure 6.
 
Effects of the electrical stimulation (ES) of the cervical sympathetic trunk on (A) the mean discharge rate of cold-sensory fibers (n = 4); (B) local blood flow (n = 6); and (C) temperature in the subscleral space (n = 3). Data obtained with different stimulation frequencies (5, 10, 15, 20, and 25 Hz) have been pooled. Data are the mean ± SEM; *P < 0.05, one-way ANOVA, Dunn (A) and Dunnett (B) methods, and paired t-test (C).
Figure 6.
 
Effects of the electrical stimulation (ES) of the cervical sympathetic trunk on (A) the mean discharge rate of cold-sensory fibers (n = 4); (B) local blood flow (n = 6); and (C) temperature in the subscleral space (n = 3). Data obtained with different stimulation frequencies (5, 10, 15, 20, and 25 Hz) have been pooled. Data are the mean ± SEM; *P < 0.05, one-way ANOVA, Dunn (A) and Dunnett (B) methods, and paired t-test (C).
Figure 7.
 
Sample recordings of nerve discharge of a scleral cold receptor fiber (A), and parallel ocular blood flow and scleral temperature changes (B) during electrical stimulation of the cervical sympathetic nerve at 20 Hz. Horizontal bars: stimulus duration.
Figure 7.
 
Sample recordings of nerve discharge of a scleral cold receptor fiber (A), and parallel ocular blood flow and scleral temperature changes (B) during electrical stimulation of the cervical sympathetic nerve at 20 Hz. Horizontal bars: stimulus duration.
Figure 8.
 
Average change in temperature measured in the subscleral space when the sclera was covered by lids and orbit (○) or exposed (•). (A) During electrical stimulation of the sympathetic nerve (ES) at 20 Hz (n = 7). (B) During carotid artery occlusion (CO; n = 5). Data are the mean ± SEM.
Figure 8.
 
Average change in temperature measured in the subscleral space when the sclera was covered by lids and orbit (○) or exposed (•). (A) During electrical stimulation of the sympathetic nerve (ES) at 20 Hz (n = 7). (B) During carotid artery occlusion (CO; n = 5). Data are the mean ± SEM.
Figure 9.
 
Effects of carotid occlusion (CO) on (A) the mean discharge rate of scleral cold thermoreceptor fibers (n = 27), (B) local blood flow (n = 31), and (C) ocular temperature (n = 3). Data are mean ± the SEM; *P < 0.05 one-way ANOVA, Dunn method (A, B) and paired t-test (C).
Figure 9.
 
Effects of carotid occlusion (CO) on (A) the mean discharge rate of scleral cold thermoreceptor fibers (n = 27), (B) local blood flow (n = 31), and (C) ocular temperature (n = 3). Data are mean ± the SEM; *P < 0.05 one-way ANOVA, Dunn method (A, B) and paired t-test (C).
Figure 10.
 
Sample recordings of (A) impulse discharge (frequency/sec) of a scleral cold receptor and (B) local blood flow and scleral temperature changes recorded simultaneously during a 30-second carotid artery occlusion. Horizontal bar: duration of the occlusion.
Figure 10.
 
Sample recordings of (A) impulse discharge (frequency/sec) of a scleral cold receptor and (B) local blood flow and scleral temperature changes recorded simultaneously during a 30-second carotid artery occlusion. Horizontal bar: duration of the occlusion.
The authors thank Alfonso Pérez-Vegara and Simón Moya for technical assistance and also Adolfo Aracil for his contribution to a part of the experiments. 
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Figure 1.
 
Distribution of cold thermoreceptors on the scleral surface of a feline eye. (A) Location of the receptive fields of scleral and limbal cold thermoreceptor fibers in the temporal side of the eye. (B) Front view of the feline eye illustrates that the sclera and a large part of the limbus are not exposed to external temperature changes.
Figure 1.
 
Distribution of cold thermoreceptors on the scleral surface of a feline eye. (A) Location of the receptive fields of scleral and limbal cold thermoreceptor fibers in the temporal side of the eye. (B) Front view of the feline eye illustrates that the sclera and a large part of the limbus are not exposed to external temperature changes.
Figure 2.
 
Sample recordings of impulse frequency of scleral cold receptor fibers. (A) Traces are examples of the spontaneous activity of two different cold fibers at 32°C. (B) Impulse discharge of a cold unit in response to a cooling pulse from an adapting temperature of 35°C down to 21°C (bottom trace).
Figure 2.
 
Sample recordings of impulse frequency of scleral cold receptor fibers. (A) Traces are examples of the spontaneous activity of two different cold fibers at 32°C. (B) Impulse discharge of a cold unit in response to a cooling pulse from an adapting temperature of 35°C down to 21°C (bottom trace).
Figure 3.
 
Response of a scleral cold thermoreceptor to −2°C, −4°C, and −6°C cooling pulses applied sequentially with a 2-minute interval from an adapting temperature of 35°C (top record). (A) Instantaneous frequency during a 30-second period, including the dynamic and static phases of the cooling pulse (bottom trace). (B) Interval histogram showing the distribution of the intervals between nerve impulses. Note the shift to the left with larger cooling pulses.
Figure 3.
 
Response of a scleral cold thermoreceptor to −2°C, −4°C, and −6°C cooling pulses applied sequentially with a 2-minute interval from an adapting temperature of 35°C (top record). (A) Instantaneous frequency during a 30-second period, including the dynamic and static phases of the cooling pulse (bottom trace). (B) Interval histogram showing the distribution of the intervals between nerve impulses. Note the shift to the left with larger cooling pulses.
Figure 4.
 
Scleral cold thermoreceptor response to cold stimulation. (A) Average response of 11 cold-sensory fibers during the dynamic (○) and static (•) components of the cooling pulse. Data are the mean ± SEM of the mean discharge rate. (B) Stimulus-response curve obtained from fibers shown in (A) during the dynamic change of scleral temperature between 35°C and 34°C. The firing frequency (impulses/second) was linearly related to the temperature reduction (r 2 = 0.74). Inset: relationship between stimulus temperature measured at the tip of the probe (abscissa) and temperature measured inside the sclera (ordinate) during cooling pulses (r 2 = 0.96), to illustrate the correlation between temperature reduction in the sclera and the probe tip during the dynamic component of the cooling pulse.
Figure 4.
 
Scleral cold thermoreceptor response to cold stimulation. (A) Average response of 11 cold-sensory fibers during the dynamic (○) and static (•) components of the cooling pulse. Data are the mean ± SEM of the mean discharge rate. (B) Stimulus-response curve obtained from fibers shown in (A) during the dynamic change of scleral temperature between 35°C and 34°C. The firing frequency (impulses/second) was linearly related to the temperature reduction (r 2 = 0.74). Inset: relationship between stimulus temperature measured at the tip of the probe (abscissa) and temperature measured inside the sclera (ordinate) during cooling pulses (r 2 = 0.96), to illustrate the correlation between temperature reduction in the sclera and the probe tip during the dynamic component of the cooling pulse.
Figure 5.
 
Organization of burst discharges of scleral cold thermoreceptors at different static temperatures. The number of impulses per burst (A), the burst duration (B), and the interburst interval (C) were represented versus steady temperature. Data are mean ± SEM, n = 4.
Figure 5.
 
Organization of burst discharges of scleral cold thermoreceptors at different static temperatures. The number of impulses per burst (A), the burst duration (B), and the interburst interval (C) were represented versus steady temperature. Data are mean ± SEM, n = 4.
Figure 6.
 
Effects of the electrical stimulation (ES) of the cervical sympathetic trunk on (A) the mean discharge rate of cold-sensory fibers (n = 4); (B) local blood flow (n = 6); and (C) temperature in the subscleral space (n = 3). Data obtained with different stimulation frequencies (5, 10, 15, 20, and 25 Hz) have been pooled. Data are the mean ± SEM; *P < 0.05, one-way ANOVA, Dunn (A) and Dunnett (B) methods, and paired t-test (C).
Figure 6.
 
Effects of the electrical stimulation (ES) of the cervical sympathetic trunk on (A) the mean discharge rate of cold-sensory fibers (n = 4); (B) local blood flow (n = 6); and (C) temperature in the subscleral space (n = 3). Data obtained with different stimulation frequencies (5, 10, 15, 20, and 25 Hz) have been pooled. Data are the mean ± SEM; *P < 0.05, one-way ANOVA, Dunn (A) and Dunnett (B) methods, and paired t-test (C).
Figure 7.
 
Sample recordings of nerve discharge of a scleral cold receptor fiber (A), and parallel ocular blood flow and scleral temperature changes (B) during electrical stimulation of the cervical sympathetic nerve at 20 Hz. Horizontal bars: stimulus duration.
Figure 7.
 
Sample recordings of nerve discharge of a scleral cold receptor fiber (A), and parallel ocular blood flow and scleral temperature changes (B) during electrical stimulation of the cervical sympathetic nerve at 20 Hz. Horizontal bars: stimulus duration.
Figure 8.
 
Average change in temperature measured in the subscleral space when the sclera was covered by lids and orbit (○) or exposed (•). (A) During electrical stimulation of the sympathetic nerve (ES) at 20 Hz (n = 7). (B) During carotid artery occlusion (CO; n = 5). Data are the mean ± SEM.
Figure 8.
 
Average change in temperature measured in the subscleral space when the sclera was covered by lids and orbit (○) or exposed (•). (A) During electrical stimulation of the sympathetic nerve (ES) at 20 Hz (n = 7). (B) During carotid artery occlusion (CO; n = 5). Data are the mean ± SEM.
Figure 9.
 
Effects of carotid occlusion (CO) on (A) the mean discharge rate of scleral cold thermoreceptor fibers (n = 27), (B) local blood flow (n = 31), and (C) ocular temperature (n = 3). Data are mean ± the SEM; *P < 0.05 one-way ANOVA, Dunn method (A, B) and paired t-test (C).
Figure 9.
 
Effects of carotid occlusion (CO) on (A) the mean discharge rate of scleral cold thermoreceptor fibers (n = 27), (B) local blood flow (n = 31), and (C) ocular temperature (n = 3). Data are mean ± the SEM; *P < 0.05 one-way ANOVA, Dunn method (A, B) and paired t-test (C).
Figure 10.
 
Sample recordings of (A) impulse discharge (frequency/sec) of a scleral cold receptor and (B) local blood flow and scleral temperature changes recorded simultaneously during a 30-second carotid artery occlusion. Horizontal bar: duration of the occlusion.
Figure 10.
 
Sample recordings of (A) impulse discharge (frequency/sec) of a scleral cold receptor and (B) local blood flow and scleral temperature changes recorded simultaneously during a 30-second carotid artery occlusion. Horizontal bar: duration of the occlusion.
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