Modification of cornea-evoked reflex blinks in rats

Although maintaining the tear film on the cornea is the most important role of blinking, information about the organization and modification of cornea-evoked blinks is sparse. This study characterizes cornea-evoked blinks and their modification in urethane-anesthetized rats. Cornea-evoked blinks typically begin 16.2 ms after an electrical stimulus to the cornea and last an average of 50.2 ms. In anesthetized rats, the blink only occurs ipsilateral to the stimulus. In response to cornea stimulation, the orbicularis oculi EMG activity typically exhibits two bursts that correlate with the arrival of A and C-fiber inputs to the spinal trigeminal complex. In the paired-stimulus paradigm, suppression of the blink evoked by the second cornea stimulus occurs for interstimulus intervals less than 300 ms and is exclusively unilateral. Stimulation of the contralateral cornea does not affect subsequent blinks evoked from stimulation of the ipsilateral cornea. To determine whether activation of cornea-related neurons in the border region between the spinal trigeminal caudalis subdivision and the C1 spinal cord (Vc/C1) inhibits the second blink in the paired-stimulus paradigm, we examine the suppression of cornea-evoked blinks caused by microstimulation in this region. This suppression of orbicularis oculi EMG activity begins 8.3 ms after Vc/C1 stimulation. Activation of this region, however, is unlike suppression in the paired-stimulus paradigm because Vc/C1 activation bilaterally inhibits cornea-evoked blinks. Thus, activation of Vc/C1 is a previously unidentified mechanism for modulating cornea-evoked blinks.     

GABA(A) receptor activation modulates corneal unit activity in rostral and caudal portions of trigeminal subnucleus caudalis

Corneal nociceptors terminate at the trigeminal subnucleus interpolaris/caudalis (Vi/Vc) transition and subnucleus caudalis/upper cervical spinal cord (Vc/C1) junction regions of the lower brain stem. The aims of this study were to determine if local GABAA receptor activation modifies corneal input to second-order neurons at these regions and if GABAA receptor activation in one region affects corneal input to the other region. In barbiturate-anesthetized male rats, corneal nociceptors were excited by pulses of CO2 gas, and GABAA receptors were activated by microinjections of the selective agonist muscimol. Local muscimol injection at the site of recording inhibited all Vi/Vc and Vc/C1 units tested and was reversed partially by bicuculline. To test for ascending intersubnuclear communication, muscimol injection into the caudal Vc/C1 junction, remote from the recording site at the Vi/Vc transition, inhibited the evoked response of most corneal units, although some neurons were enhanced. Injection of the nonselective synaptic blocking agent, CoCl2, remotely into the Vc/C1 region inhibited the evoked response of all Vi/Vc units tested. To test for descending intersubnuclear communication, muscimol was injected remotely into the rostral Vi/Vc transition and enhanced the evoked activity of all corneal units tested at the caudal Vc/C1 junction. These results suggest that GABAA receptor mechanisms play a significant role in corneal nociceptive processing by second-order trigeminal brain stem neurons. GABAA receptor mechanisms act locally at both the Vi/Vc transition and Vc/C1 junction regions to inhibit corneal input and act through polysynaptic pathways to modify corneal input at multiple levels of the trigeminal brain stem complex.     

Responses of medullary dorsal horn neurons to corneal stimulation by CO(2) pulses in the rat

Corneal-responsive neurons were recorded extracellularly in two regions of the spinal trigeminal nucleus, subnucleus interpolaris/caudalis (Vi/Vc) and subnucleus caudalis/upper cervical cord (Vc/C1) transition regions, from methohexital-anesthetized male rats. Thirty-nine Vi/Vc and 26 Vc/C1 neurons that responded to mechanical and electrical stimulation of the cornea were examined for convergent cutaneous receptive fields, responses to natural stimulation of the corneal surface by CO(2) pulses (0, 30, 60, 80, and 95%), effects of morphine, and projections to the contralateral thalamus. Forty-six percent of mechanically sensitive Vi/Vc neurons and 58% of Vc/C1 neurons were excited by CO(2) stimulation. The evoked activity of most cells occurred at 60% CO(2) after a delay of 7-22 s. At the Vi/Vc transition three response patterns were seen. Type I cells (n = 11) displayed an increase in activity with increasing CO(2) concentration. Type II cells (n = 7) displayed a biphasic response, an initial inhibition followed by excitation in which the magnitude of the excitatory phase was dependent on CO(2) concentration. A third category of Vi/Vc cells (type III, n = 3) responded to CO(2) pulses only after morphine administration (>1.0 mg/kg). At the Vc/C1 transition, all CO(2)-responsive cells (n = 15) displayed an increase in firing rates with greater CO(2) concentration, similar to the pattern of type I Vi/Vc cells. Comparisons of the effects of CO(2) pulses on Vi/Vc type I units, Vi/Vc type II units, and Vc/C1 corneal units revealed no significant differences in threshold intensity, stimulus encoding, or latency to sustained firing. Morphine (0.5-3.5 mg/kg iv) enhanced the CO(2)-evoked activity of 50% of Vi/Vc neurons tested, whereas all Vc/C1 cells were inhibited in a dose-dependent, naloxone-reversible manner. Stimulation of the contralateral posterior thalamic nucleus antidromically activated 37% of Vc/C1 corneal units; however, no effective sites were found within the ventral posteromedial thalamic nucleus or nucleus submedius. None of the Vi/Vc corneal units tested were antidromically activated from sites within these thalamic regions. Corneal-responsive neurons in the Vi/Vc and Vc/C1 regions likely serve different functions in ocular nociception, a conclusion reflected more by the difference in sensitivity to analgesic drugs and efferent projection targets than by the CO(2) stimulus intensity encoding functions. Collectively, the properties of Vc/C1 corneal neurons were consistent with a role in the sensory-discriminative aspects of ocular pain due to chemical irritation. The unique and heterogeneous properties of Vi/Vc corneal neurons suggested involvement in more specialized ocular functions such as reflex control of tear formation or eye blinks or recruitment of antinociceptive control pathways.     

Endotoxin-induced uveitis causes long-term changes in trigeminal subnucleus caudalis neurons

Endotoxin-induced uveitis (EIU) is commonly used in animals to mimic ocular inflammation in humans. Although the peripheral aspects of EIU have been well studied, little is known of the central neural effects of anterior eye inflammation. EIU was induced in male rats by endotoxin or lipopolysaccharide (LPS, 1 mg/kg ip) given 2 or 7 days earlier. Neurons responsive to mechanical stimulation of the ocular surface were recorded under barbiturate anesthesia at the trigeminal subnucleus interpolaris/caudalis (Vi/Vc) transition and subnucleus caudalis/cervical cord (Vc/C1) junction, the main terminal regions for corneal nociceptors. Two days after LPS, Vc/C1 units had reduced responses to histamine, nicotine, and CO₂ gas applied to the ocular surface, whereas unit responses were increased 7 days after LPS. Those units with convergent cutaneous receptive fields at Vc/C1 were enlarged 7 days after LPS. Units at the Vi/Vc transition also had reduced responses to histamine and CO₂ 2 days after LPS but no enhancement was seen at 7 days. Tear volume evoked by CO₂ was reduced 2 days after LPS and returned toward control values by 7 days, whereas CO₂-evoked eye blinks were normal at 2 days and increased 7 days after LPS. These results indicate that a single exposure to endotoxin causes long-term changes in the excitability of second-order neurons responsive to noxious ocular stimulation. The differential effects of EIU on tear volume and eye blink lend further support for the hypothesis that ocular-sensitive neurons at the Vi/Vc transition and Vc/C1 junction regions mediate different aspects of pain during intraocular inflammation.     

Ocular surface-evoked Fos-like immunoreactivity is enhanced in trigeminal subnucleus caudalis by prior exposure to endotoxin

Endotoxin-induced uveitis (EIU) is a common animal model for anterior uveitis in humans that causes long-term changes in trigeminal brain stem neurons. This study used c-fos immunohistochemistry to assess the effects of different routes of administration of endotoxin on activation of trigeminal brain stem neurons produced by ocular surface stimulation. A single dose of endotoxin (lipopolysaccharide (LPS)) given to male rats by systemic (i.p., 1 mg/kg) or intraocular (ivt, 20 μg) routes increased the number of Fos-positive neurons in rostral (trigeminal subnucleus interpolaris/subnucleus transition (Vi/Vc)) and caudal portions of trigeminal subnucleus caudalis (trigeminal subnucleus caudalis/upper cervical spinal cord transition (Vc/C_1–2)) by 20% mustard oil (MO) applied to the ocular surface 7 days, but not at 2 days, after LPS compared with naïve rats. I.c.v. (20 μg) LPS did not affect MO-evoked Fos. To determine if the pattern of enhanced Fos expression after systemic LPS also depended on the nature of the ocular surface stimulus, additional groups received ocular stimulation by 10% histamine or dry eye conditions. Seven days, but not 2 days, after i.p. LPS both histamine- and dry eye–evoked Fos was increased at the Vi/Vc transition, while smaller effects were seen at other regions. These results suggested that EIU modulation of trigeminal brain stem neuron activity was mediated mainly by peripheral actions of LPS. Enhancement of Fos at the Vi/Vc region after MO, histamine and dry eye conditions supports the hypothesis that this region integrates innocuous as well as noxious sensory information, while more caudal portions of Vc process mainly nociceptive signals from the eye.     

Capsaicin-responsive corneal afferents do not contain TRPV1 at their central terminals in trigeminal nucleus caudalis in rats

We examined the substrates for ocular nociception in adult male Sprague-Dawley rats. Capsaicin application to the ocular surface in awake rats evoked nocifensive responses and suppressed spontaneous grooming responses. Thus, peripheral capsaicin was able to activate the central pathways encoding ocular nociception. Our capsaicin stimulus evoked c-Fos expression in a select population of neurons within rostral trigeminal nucleus caudalis in anesthetized rats. These activated neurons also received direct contacts from corneal afferent fibers traced with cholera toxin B from the corneal surface. However, the central terminals of the corneal afferents that contacted capsaicin-activated trigeminal neurons did not contain TRPV1. To determine if TRPV1 expression had been altered by capsaicin stimulation, we examined TRPV1 content of corneal afferents in animals that did not receive capsaicin stimulation. These studies confirmed that while TRPV1 was present in 30% of CTb-labeled corneal afferent neurons within the trigeminal ganglion, TRPV1 was only detected in 2% of the central terminals of these corneal afferents within the trigeminal nucleus caudalis. Other TRP channels were also present in low proportions of central corneal afferent terminals in unstimulated animals (TRPM8, 2%; TRPA1, 10%). These findings indicate that a pathway from the cornea to rostral trigeminal nucleus caudalis is involved in corneal nociceptive transmission, but that central TRP channel expression is unrelated to the type of stimulus transduced by the peripheral nociceptive endings.     

The trigeminally evoked blink reflex

In this study, we characterized the pathways that generate the trigeminal blink reflex in the guinea pig. Blinks were evoked by stimulation of the supraorbital branch of the trigeminal nerve and measured by recording electromyographic activity in the lid-closing orbicularis oculi muscle (OOemg) and, in one case, lid position. Blinks evoked by stimulation of the supraorbital nerve consisted of two bursts of muscle activity ipsilateral to the side of stimulation. The first, R1, had a latency of 6.9 ms and the second, R2, had a latency of 17.25 ms. Increasing stimulus intensity to 3 times threshold for evoking an ipsilateral blink elicited an R1 and R2 response contralaterally, with latencies of 9.2 ms and 19.25 ms, respectively. We investigated the causes for this bipartite response that is seen in the guinea pig, as well as other mammals including humans. The two-component response could arise from different populations of afferents, or from different central circuits, or a combination of these two causes. Multiunit recording in the trigeminal ganglion and simultaneous measurement of the OOemg showed that activation of A afferents alone was sufficient to elicit both the R1 and the R2 responses, but that activation of A afferents could enhance both responses. Different neural circuits, however, produce the R1 and R2 responses. Transganglionic tracing with wheatgerm agglutin or choleragenoid subunit of cholera toxin bound to HRP revealed that primary afferents from the supraorbital branch of the trigeminal nerve terminated densely in the dorsal horn of spinal cord segment C1 and in the caudalis-interpolaris border region of the spinal trigeminal nucleus. Injections of HRP into the orbicularis oculi motoneuron region of the facial nucleus showed that both of these regions projected to the facial nucleus. Hemisections at the level of C1 eliminated the R2 blink response, but not the R1 response, evoked by stimulation of the supraorbital branch of the trigeminal nerve. Subsequent hemisections at the level of the obex eliminated the R1 response. Microinjections of the GABAB agonist baclofen into the spinal trigeminal nucleus at the level of the obex abolished the R1 but not the R2 response. Thus, the spinal trigeminal nucleus produces the R1 component, whereas the R2 component originates in the C1 region of the spinal cord.     

Behavioral and neurophysiological correlates of nociception in an animal model of photokeratitis

Ocular exposure to ultraviolet irradiation (UVR) induces photokeratitis, a common environmental concern that inflames ocular tissues and causes pain. The central neural mechanisms that contribute to the sensory aspects of photokeratitis after UVR are not known. In awake male rats, ocular surface application of hypertonic saline evoked eye wipe behavior that was enhanced 2–3 days after UVR and returned to control levels by 7 days. Similarly, under isoflurane anesthesia, hypertonic saline-evoked activity of ocular neurons in superficial laminae at the trigeminal subnucleus caudalis/cervical (Vc/C1) region was enhanced 2 days, but not 7 days, after UVR. By contrast, the response of neurons at the interpolaris/caudalis (Vi/Vc) transition region to hypertonic saline was not affected by UVR. The background activity and convergent cutaneous receptive field areas of Vc/C1 or Vi/Vc neurons were not affected by UVR. Aqueous humor protein levels were elevated 2 and 7 days after UVR. UVR enhanced nociceptive behavior, after a latent period, with a time course similar to that of ocular neurons in superficial laminae at the Vc/C1 region. The Vc/C1 region plays a key role in primary hyperalgesia induced by UVR, whereas the Vi/Vc region likely mediates other aspects of ocular function.     

Responses of feline trigeminal spinal tract nucleus neurons to stimulation of the middle meningeal artery and sagittal sinus

1. Extracellular single-unit activity was recorded from 250 trigeminal (V) spinal tract nucleus neurons that were excited by electrical stimulation of the middle meningeal artery (MMA) and/or sagittal sinus (SS) in anesthetized cats. One hundred and thirty of these neurons were located in the V subnucleus caudalis (Vc), and the remaining 120 neurons were located in the V subnucleus oralis (Vo) or rostral part of the V subnucleus interpolaris (Vi). In many cases these neurons were also examined for the existence of orofacial receptive fields (RFs) by applying mechanical stimuli to the orofacial region. 2. The mean minimum latencies to suprathreshold electrical stimulation of the MMA and the SS were similar for Vc and Vo/Vi neurons. Excitation of Vc neurons occurred at latencies of 14.3 +/- 1.0 (n = 102) and 17.4 +/- 1.6 ms (n = 36) to MMA and SS stimulation, respectively. Excitation of Vo/Vi neurons occurred at latencies of 12.4 +/- 0.9 (n = 86) and 16.4 +/- 1.1 ms (n = 58) to MMA and SS stimulation, respectively. These latencies correspond to mean conduction velocities of approximately 5.2 and 4.0 m/s to MMA and SS stimulation, respectively. 3. Mechanical stimulation of the vessels evoked neuronal responses in five of eight MMA-activated neurons tested and three of five SS-activated neurons tested. 4. Almost all of the neurons tested (127 of 131) had peripheral RFs, and all were in the orofacial region. Nearly all (95%) Vc neurons had RFs within or including the ophthalmic facial region. The RFs of most (67%) Vo/Vi neurons also had RFs within or including ophthalmic regions, but in some cases were only within maxillary and/or mandibular regions. 5. Most of the Vc neurons (87%) were classified on the basis of their cutaneous inputs as nociceptive. The incidence of nociceptive neurons in Vo/Vi was also high (61%), although less than in Vc. In both the Vc and Vo/Vi neuronal populations, MMA-activated neurons were more likely to have nociceptive peripheral inputs than SS-activated neurons. 6. Histological reconstruction of recording sites indicated that the MMA- and/or SS-activated Vc neurons were concentrated in the lateral half of laminae III-V. The responsive neurons at the level of Vo/rostral Vi were not confined to any particular portion of these subnuclei. 7. These findings indicate that sensory afferents that innervate the dural arteries and venous sinuses are capable of activating neurons throughout the V spinal tract nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)     

The distribution of primary afferent terminals from the eyelids of macaque monkeys

 Trigeminal sensory afferents from the eyelids convey two types of information that are important for the blink reflex. Movement of the lashes activates low-threshold mechanoreceptors which evoke protective blinks. Information about eyelid position is also transmitted centrally and is used to adapt the metrics of the blink reflex to changing conditions over time. This study employed transganglionic transport of horseradish peroxidase conjugated to choleratoxin-B subunit or wheat-germ agglutinin to investigate trigeminal afferents supplying the eyelids of macaque monkeys. Ganglion cells labeled from upper- and lower-lid injections were located in the ophthalmic and maxillary portions of the trigeminal ganglion, respectively. In both cases, labeled terminals were observed ipsilateral to the injected eyelid in the principal and spinal trigeminal nuclei. However, only a few labeled terminals were present in the principal nucleus, and very sparse terminal labeling was confined to a few locations along the ventral border of the pars oralis and interpolaris of the spinal trigeminal nucleus. The main concentration of label was found in the pars caudalis at and immediately below the spinomedullary junction. The terminal field from the upper eyelid was located ventrally in the pars caudalis, and that from the lower eyelid was located more dorsally. In both cases, the labeled terminal field was densest within lamina II of the spinal trigeminal nucleus. The heavy concentration of eyelid central terminals at the spinomedullary junction is surprising in light of physiological studies indicating representation of all parts of the face throughout the trigeminal nucleus. The distribution of eyelid afferent terminals in the macaque is caudal to the main concentration of corneal afferent terminals at the pars interpolaris/caudalis border. This may be a basis for differences seen in blinks produced by corneal as opposed to supraorbital stimulation. The presence of a single major site of eyelid primary afferent terminals suggests that sensory input for both eyelid proprioception and blink-reflex activation passes through this segment of the spinal trigeminal nucleus. These results provide a basis for investigation of the central connections of pars caudalis neurons in order to better establish the pathways producing trigeminally evoked blinks and blink adaptation. Received: 6 April 1998 / Accepted: 9 June 1998     

Activation of central 5HT2A receptors reduces the craniofacial nociception of rats

We assessed the contribution of central 5HT2A receptors to the craniofacial tissue nociception in naïve male rats. First, we tested whether activation of central 5HT2A receptors affected nociceptive neural activities recorded from superficial laminae of the trigeminal subnucleus caudalis (Vc)/upper cervical spinal cord junction (Vc/C2) region. Two types of units, such as deep-nociceptive or skin–wide dynamic range (WDR) units were identified from extracellular recordings. Topical administration of 5HT2A receptor agonist, (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) onto the Vc/C2 region significantly reduced deep-nociceptive unit discharges evoked by formalin injection into the masseter muscle. Noxious pinch stimulation to the facial skin-evoked skin–WDR unit discharges was significantly reduced by topical administration of 0.1 mg/rat DOI onto the Vc/C2 region. Second, we tested whether i.c.v. administration of DOI affected Fos-like immunoreactivity (-LI) evoked by formalin injection into the masseter muscle. Fos-LI was significantly induced mainly at the ventrolateral (vl) area of trigeminal subnucleus interpolaris (Vi)/Vc junction (vl-Vi/Vc) region and Vc/C2 region in vehicle-treated rats. Formalin-evoked Fos-LI was significantly reduced in laminae I–II of the Vc/C2, but not vl-Vi/Vc region after i.c.v. administration of DOI. Finally, orofacial nocifensive behavioral activities evoked by formalin injection into the masseter muscle were significantly reduced by intracisternal administration of DOI. These results suggest that 5HT2A receptors in the Vc/C2 region mediate antinociceptive effects in the craniofacial nociception.     

Suppression of visual cortical activity following tactile periorbital stimulation; its role during eye blinks

Summary Single units were recorded in area 17 of anesthetized and paralyzed cats. The discharges of cortical units, either spontaneous or driven by two dimensional visual drifting noise, were analyzed during unilateral tactile stimulation (air puffs or taps) of the skin around the eye. This stimulation evokes a blink response in the normal non-curarized animal. The activity in the branch of the facial nerve innervating the orbicularis oculi muscles responsible for the blink was also recorded on the stimulated side. Following the mechanical stimulation, the discharges of both simple and complex visual cells were strongly inhibited with a latency of 70–80 ms. Inhibition was sometimes preceded by a brief increase in firing rate. This typical response was present only when the cutaneous stimulus was effective in triggering a discharge in the motor nerve which drives the orbicularis oculi muscles. Moreover, when a visual response was evoked by a temporary masking of the visual stimulus, this response was suppressed by the association with a tap stimulation.Supported by the DGRST grant (DN 81.E.1489) to P.B.     

Bright light produces Fos-positive neurons in caudal trigeminal brainstem

Excessive discomfort after exposure to bright light often occurs after ocular injury and during headache. Although the trigeminal nerve is necessary for light-evoked discomfort, the mechanisms underlying this phenomenon, often referred to generally as photophobia, are not well defined. Quantitative Fos-like immunoreactivity (Fos-LI) was used to determine the pattern of neuronal activation in the caudal brainstem after bright light stimulation and, secondly, whether a neurovascular mechanism within the eye contributes to this response. Under barbiturate anesthesia, male rats were exposed to low (1×10⁴ lx) or high intensity (2×10⁴ lx) light delivered from a thermal neutral source for 30 min (30 s ON, 30 s OFF) and allowed to survive for 90 min. Intensity-dependent increases in Fos-LI were seen in laminae I–II at the trigeminal caudalis/cervical cord junction region (Vc/C1) and nucleus tractus solitarius (NTS). Fos-LI also increased at the trigeminal interpolaris/caudalis transition (Vi/Vc_vl) and dorsal paratrigeminal (dPa5) regions independent of intensity. Intravitreal injection of norepinephrine greatly reduced light-evoked Fos-LI at the Vc/C1, dPa5 and NTS, but not at the Vi/Vc transition. Lidocaine applied to the ocular surface had no effect on Fos-LI produced in trigeminal brainstem regions. These results suggested that multiple regions of the caudal trigeminal brainstem complex integrate light-related sensory information. Fos-LI produced at the dPa5 and NTS, coupled with norepinephrine-induced inhibition, was consistent with the hypothesis that light-evoked activation of trigeminal brainstem neurons involves an intraocular neurovascular mechanism with little contribution from neurons that supply the ocular surface.     

Neural pathways mediating the corneal blink reflex and Bell's phenomenon in the cat

Two central projections from the corneal representation of the sensory trigeminal complex in the cat were demonstrated with horseradish peroxidase, autoradiographic and Golgi methods: (1) to the dorsal subdivision of the ipsilateral facial nucleus that innervates the orbicularis oculi muscle; and (2) to the bordering area between the contralateral central gray matter and the oculomotor nucleus, which receives dendrites of the oculomotor cells innervating the contralateral superior rectus muscle. These two routes probably mediate early responses of the corneal blink reflex and Bell's phenomenon, respectively.     

GABAA receptor-mediated tonic currents in substantia gelatinosa neurons of rat spinal trigeminal nucleus pars caudalis

In the present study, we describe GABAA receptor-mediated tonic inhibitory currents in the substantia gelatinosa (SG) region of rat spinal trigeminal nucleus pars caudalis (Vc). The GABA(A) receptor-mediated tonic currents were identified by bath-application of the GABAA receptor antagonists, picrotoxin (1mM), SR95531 (100microM) and bicuculline (100microM). All three antagonists completely blocked outward spontaneous (phasic) inhibitory postsynaptic currents, but only picrotoxin and bicuculline induced a significant (>5pA) inward shift of holding currents at a holding potential (Vh) of 0mV in 60-70% of SG neurons, revealing the existence of tonic outward currents. The tonic currents were resistant to further the blockades of glycine receptors or those in addition to glutamate receptors and voltage-dependent sodium channels. An acute bath-application of THDOC (0.1microM), the stress-related neurosteroid, did enhance tonic currents, but only in a small population of SG neurons. In addition, slices incubated with THDOC for 30min increased the probability of neurons with significant tonic currents. The GABAergic tonic inhibition demonstrated in this study may play a significant role in the sensory processing system of the Vc.     

Ultraviolet irradiation of the eye and Fos-positive neurons induced in trigeminal brainstem after intravitreal or ocular surface transient receptor potential vanilloid 1 activation

The interior structures of the eye are well supplied by the trigeminal nerve; however, the function of these afferent fibers is not well defined. The aim of this study was to use c-fos like immunohistochemistry (Fos-LI) to map the trigeminal brainstem complex after intravitreal microinjection or ocular surface application of capsaicin, a selective transient receptor potential vanilloid 1 (TRPV1) agonist in male rats under barbiturate anesthesia. The effect of ocular inflammation on Fos-LI was tested 2 or 7 days after UV irradiation of the eye. In non-inflamed controls, intravitreal capsaicin produced peaks of Fos-LI at the trigeminal subnucleus interpolaris/caudalis (Vi/Vcvl) transition and in superficial laminae at the caudalis/upper cervical cord (Vc/C1) junction regions. At the Vc/C1 junction intravitreal capsaicin induced Fos-LI in a dose-dependent manner, while at the Vi/Vcvl transition responses were similar after vehicle or capsaicin injections. Two days, but not 7 days, after UV irradiation intravitreal and ocular surface capsaicin-evoked Fos-LI at the Vc/C1 junction and nucleus tractus solitarius (NTS) were markedly enhanced, whereas the responses at the Vi/Vcvl transition were not different from non-inflamed controls. More than 80% of trigeminal ganglion neurons labeled after intravitreal microinjection of Fluorogold also expressed immunoreactivity for the TRPV1 receptor. These findings suggested that most intraocular trigeminal sensory nerves serve as nociceptors. The similar pattern and magnitude of Fos-LI after capsaicin suggested that TRPV1-responsive trigeminal nerves that supply intraocular and ocular surface tissues form a unified integrative circuit in the caudal brainstem. Intensity coding of capsaicin concentration and facilitation of Fos-LI expression after UV irradiation strongly supported the hypothesis that the Vc/C1 junction was critical for nociceptive processing related to ocular pain, whereas the Vi/Vcvl transition region likely served other functions in ocular homeostasis under naïve and inflamed conditions.     

Midbrain 6-hydroxydopamine lesions modulate blink reflex excitability

The blink reflex abnormalities present in the 6 hydroxydopamine (6-OHDA) lesioned rat model of parkinsonism mimicked those of the human with Parkinon's disease. In alert rats, we monitored the long and short latency components of the orbicularis oculi electromyographic (OOemg) response evoked by electrical stimulation of the supraorbital branch of the trigeminal nerve (SO). Two paradigms, habituation and double pulse, provided a measure of blink reflex excitability. In normal rats, repeated stimulation of the SO produced habituation of the R2 component of the blink. In the double pulse paradigm, presentation of two identical SO stimuli resulted in a reduced or suppressed OOemg response to the second stimulus relative to the first. In rats with complete, unilateral lesions of midbrain dopamine neurons, repeated SO stimulation produced facilitation rather than habituation of the R2 component of the blink reflex. This facilitation occurred only with the eyelid contralateral to the lesion. In the double pulse paradigm, the lesioned rats showed increased excitability rather than suppression. This effect occurred bilaterally, although the increased excitability was strongest contralateral to the lesion. Rats with partial lesions of midbrain dopamine neurons exhibited qualitatively similar, but less pronounced blink reflex abnormalities. The R1 component of the blink reflex was unaffected by either the complete or partial lesions. Thus, modification of the blink reflex by 6-OHDA lesions provides a reproducible parkinsonian-like symptom which is amenable to investigations of increases in reflex excitability.     

Morphology and physiology of lamina I neurons of the caudal part of the trigeminal nucleus

It has been suggested that in mammals, trigeminal lamina I neurons play a role in the processing and transmission of sensory information from the orofacial region. We investigated the physiological and morphological properties of trigeminal subnucleus caudalis (Sp5C) lamina I neurons in slices prepared from the medulla oblongata of 13- to 15-day-old postnatal rats using patch-clamp recordings and subsequent biocytin-streptavidin-Alexa labeling. Twenty-five neurons were recorded and immunohistochemically stained. The Sp5C lamina I consisted of several types of neurons which, on the basis of their responses to somatic current injection, can be classified into four groups: tonic neurons, which fired throughout the depolarizing pulse; phasic neurons, which expressed an initial burst of action potentials; delayed onset neurons, which showed a significant delay of the first action potential; and single spike neurons, characterized by only one to five action potentials at the very beginning of the depolarizing pulse even at high levels of stimulation intensity. Electrical stimulation of the spinal trigeminal tract evoked AMPA receptor-mediated excitatory postsynaptic currents (EPSC) exhibiting a strong polysynaptic component. AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSC) were characterized by a 10-90% rise time of 0.50+/-0.06 ms and a decay time constant of 2.5+/-0.5 ms. The kinetic properties of NMDA receptor-mediated EPSCs were measured at +40 mV. The 10-90% rise time was 8+/-2 ms and the deactivation time constants were 94+/-31 and 339+/-72 ms, respectively. Intracellular staining and morphological analysis revealed three groups of neurons: fusiform, pyramidal, and multipolar. Statistical analysis indicated that the electrophysiological properties and morphological characteristics are correlated. Tonic and phasic neurons were fusiform or pyramidal and delayed onset and single spike neurons were multipolar. Our results show that both the physiological and morphological properties of Sp5C lamina I neurons exhibit significant differences, indicating their specific integration in the processing and transmission of sensory information from the orofacial region.     

Heterosynaptic long-term depression of craniofacial nociception: divergent effects on pain perception and blink reflex in man

Noxious low-frequency stimulation (LFS) of presynaptic nerve fibers induces long-term depression (LTD) of synaptic transmission. In vitro studies suggest a sole homosynaptic effect. Consequently, the present study addressed the hypothesis that LTD of craniofacial nociception in man is mediated by a homosynaptic mechanism. Nociceptive supraorbital afferents were excited by electric pulses via a concentric electrode in ten healthy volunteers. The electrically evoked bilateral blink reflex (BR) was recorded from both orbicularis oculi muscles by surface electrodes. The BR was evoked in blocks of ten electric stimuli each (0.1 Hz) with an interblock interval of 8 min. Conditioning noxious LFS (1 Hz, 20 min) was applied via concentric electrode either to the same site as BR test stimuli (ipsilateral) or to the corresponding contralateral forehead area (contralateral). LFS and test stimulus intensities corresponded to about threefold the pain threshold. After three baseline stimulus blocks, either conditioning ipsilateral or contralateral LFS were applied or stimulation was interrupted for 20 min as a control task. Afterwards, test stimulation blocks were continued for 40 min. Each volunteer participated in all three sessions on different days. Noxious LFS induced LTD of the BR independently from the side of conditioning stimulation. Pain perception decreased after ipsilateral LFS but not after contralateral LFS. The bilateral effect of noxious LFS on the BR provides evidence for heterosynaptic LTD based on bilateral projections of supraorbital nerve afferents onto spinal trigeminal nuclei. The divergent effect on pain perception may be due to a preferential contralateral projection of nociceptive afferents onto reflex interneurons but not onto trigeminothalamic projection neurons.