Effects of electrical stimulation of thalamic nucleus submedius and periaqueductal gray on the visceral nociceptive responses of spinal dorsal horn neurons in the rat

Electrical stimulation of the nucleus submedius (Sm) has been shown to suppress the viscerosomatic reflex (VSR), which is evoked by colorectal distension (CRD). We have examined the effects of focal electrical stimulation (0.3 ms, 50 Hz, 100 microA, 10 s) of the Sm and the periaqueductal gray (PAG) on the excitatory responses evoked by CRD in spinal dorsal horn neurons within the L6-S1 region in the urethane-anesthetized Wistar rats. Extracellular recordings were made from 32 spinal excitatory CRD responses. All of these neurons were convergent neurons with cutaneous receptive fields. The majority of the neurons (27/32) were wide dynamic range (WDR) neurons (responding to noxious and non-noxious cutaneous stimuli) while the remaining five neurons were nociceptive specific (NS) neurons (responding only to noxious cutaneous stimuli). The effects of electrical stimulation applied to 28 sites within the Sm were assessed for spinal neurons. Electrical stimulation in seven sites within the Sm (25%) inhibited the CRD excitatory response of dorsal horn neurons, while in two sites (7%) the same stimulation yielded facilitation. Electrical stimulation in the majority of the sites in the Sm (19/28, 68%) did not affect spinal excitatory CRD responses. On the other hand, electrical stimulation of the PAG clearly inhibited 20 of 22 (90%) CRD excitatory responses. These results suggest that the majority of Sm neurons may suppress VSR activity at a supraspinal reflex center rather than via a descending inhibition of spinal visceral nociceptive transmission, as is the case for the PAG.     

Inhibition of nociceptive responses of spinal dorsal horn neurons by periaqueductal gray stimulation (author's transl)]

In order to analyze analgesic effects of periaqueductal gray stimulation, experiments were carried out in nitrous oxide anesthetized cats. Spinal dorsal horn neurons receiving C-fiber inputs from the posterior tibial and/or superficial peroneal nerves were excited by noxious heat stimulation of the ipsilateral hindpaw. The evoked spike discharges were inhibited by repetitive stimulation of the periaqueductal gray at currents 300--900 microamperemeter. Effective sites for inhibition were distributed throughout the ventral half of the periaqueductal gray bilaterally. A monotonous relationship was found between stimulus intensity (skin temperature) and number of spike discharges; As predicted by Stevens' law, the number of spike discharges psi grew as a power function: psi = K(psi--psi0)beta where psi is the stimulus intensity, psi0 is the effective threshold, and K is a slope constant of the spike discharges versus stimulus intensity curve depending upon neurons. The exponent beta was approximately 1.0. The periaqueductal gray stimulation resulted in a decrease in the slope constant kappa, without alteration either in the threshold constant psi0 or in the exponent beta.     

Altered responses of nociceptive cat lamina I spinal dorsal horn neurons after chronic sciatic neuroma formation

The activity of lumbar spinal dorsal horn lamina I neurons with afferent drive from the sciatic nerve was studied in intact cats and in cats with acute sciactic nerve transection or chronic sciatic nerve transection with neuroma formation. The majority (51 of 75) of neurons recorded in lamina I ipsilateral to a neuroma had no receptive field and could only be identified by their responses to electrical stimulation of the sciatic nerve. The remainder could be activated by the sciatic nerve, but their responses to mechanical stimulation were irregular in comparison to the stable responses of cells recorded in control animals and to the responses of cells contralateral to chronic nerve lesions. Animals with acute nerve transections demonstrated as loss sciatic nerve-innvervated cells with receptive fields except for those cells located on the lateral edge of the dorsal horn, which had normal, proximal receptive fields and response characteristics. In addition, the characteristic somatotopy of lamina I cells was not observed in some cats with chronic neuromata. The mediolateral distribution of cell types indicated that some cells had altered receptive fields following chronic nerve transection. The data presented for lamina I neurons agrees with the observation of spinal cord plasticity first presented for cat dorsal horn cells. Since there is no evidence for a redistribution of intact afferent fibers following chronic nerve transection in adult mammals, the mechanism of altered somatotopy may involved alterations in synaptic efficacy at existing synapses.     

Medial hypothalamic stimulation suppresses nociceptive spinal dorsal horn neurons but not the tail-flick reflex in the rat

This study investigated the potential analgesic effects of medial hypothalamic stimulation (HS) on a measure of nocifensive behavior (tail-flick test (TF] in awake rats, and potential inhibitory effects of identical HS on spinal dorsal horn neuronal responses to noxious skin heating in the same animals anesthetized with sodium pentobarbital. Sixty-five male Sprague-Dawley rats implanted with a bipolar stimulation electrode in histologically verified medial hypothalamic sites were tested behaviorally for TF suppression during HS (100 ms trains at 100 Hz, 3/s, 100-1100 microA) in 2-4 consecutive weekly test sessions. Thirty-three of these rats were then used in electrophysiological experiments to record responses of 36 dorsal horn units to noxious skin heating (48-54 degrees C, 10 s/2 min) of the hindfoot pad in the absence of and during HS. Behaviorally, 31/65 rats had no TF suppression at the highest HS intensity tested (1100 microA), 24/65 rats exhibited aversive behavior or motor activity which disallowed reliable TF testing, and only 10/65 rats showed TF suppression in at least one test session. In electrophysiological experiments, the heat-evoked responses of 25/36 dorsal horn units were inhibited to at least 50% of control during HS. The responses of 11 units remained at 65-100% of the control responses during HS of up to 1100 microA. In rats demonstrating TF suppression, 4/7 units were inhibited. In rats with no TF suppression, 10/15 units were inhibited, and in rats showing aversive behavior, 11/14 units were inhibited by HS. These data indicate that although HS suppresses spinal nociceptive neurons, it does not cause reliable TF suppression in unanesthetized rats and bring into question the often-held assumption that stimulation-evoked descending inhibition of spinal nociceptive neurons implies behavioral analgesia.     

The effects of sympathectomy on capsaicin-evoked fos expression of spinal dorsal horn GABAergic neurons

In various hypothalamic and adjacent brain regions we have previously found a remarkable increase in nuclear estrogen receptor staining in Alzheimer's disease (AD). In order to see whether this was a general phenomenon or rather specific for those areas that are affected by the AD process we investigated ERalpha and ERbeta expression in the arginine-vasopressin (AVP) neurons of the human dorsolateral suparoptic nucleus (dl-SON), that is the major source of plasma AVP. These neurons remain exceptionally intact in AD. Changes in ER expression were studied in relation to early Alzheimer changes (i.e. hyperphosphorylated tau) and neuronal metabolism in AD as determined by the size of the Golgi apparatus (GA) or cell size. No difference in neuronal metabolism (i.e. GA size or cell size) of AVP neurons was observed between AD and control patients and no early cytoskeletal AD alterations were found confirming the resistance of the dl-SON to AD. While no differences between AD and control patients were present for ERalpha and ERbeta staining except for a lower proportion of nuclear ERbeta AVP-positive neurons in AD subjects, complex sex differences not directly related to AD were observed within each group. The main finding of the present study is that in the dl-SON, that remains active and spared of AD changes, the increase in nuclear ERs seen in adjacent affected areas in AD patients does not occur. This indicates that a rise of nuclear ERs is not a generally occurring phenomenon but rather related to the pathogenetic alterations of the AD process.     

Modulation of astroglial glutamine synthetase activity affects nociceptive behaviour and central sensitization of medullary dorsal horn nociceptive neurons in a rat model of chronic pulpitis

Previous studies indicate that the astroglial glutamate-glutamine shuttle may be involved in acute pulpal inflammatory pain by influencing central sensitization induced in nociceptive neurons in the trigeminal subnucleus caudalis [the medullary dorsal horn (MDH)] by application of an inflammatory irritant to the rat tooth pulp. The aim of this study was to test if intrathecal application to the rat medulla of the astroglial glutamine synthetase inhibitor methionine sulfoximine (MSO) can influence the central sensitization of MDH nociceptive neurons and the animal's associated behaviour that are manifested in a model of chronic pulpitis pain induced by exposure of a mandibular molar pulp. This model was found to be associated with nocifensive behaviour and enhanced reflex activity evoked by mechanical stimulation of the rat's facial skin and with immunocytochemical evidence of astroglial activation in the MDH. These features were apparent for up to 28 days post-operatively. During this post-operative period, the nocifensive behaviour and enhanced reflex activity were significantly attenuated by intrathecal application of MSO (5 μL, 10 mM) but not by vehicle application. In electrophysiological recordings of nociceptive neuronal activity in the MDH, central sensitization was also evident in pulp-exposed rats but not in intact rats and could be significantly attenuated by MSO application but not by vehicle application. These behavioural and neuronal findings suggest that the astroglial glutamate-glutamine shuttle is responsible for the maintenance of inflammation-induced nocifensive behavioural changes and the accompanying central sensitization in MDH nociceptive neurons in this chronic pulpitis pain model.     

Inhibition of rat spinal cord dorsal horn neurons by non-segmental, noxious cutaneous stimuli

Extracellular single unit recordings were obtained from spinal cord dorsal horn neurons in halothane-anesthetized rats. Inhibitory effects induced by noxious mechanical or electrical stimuli applied to a remote area of the body surface were assessed on the spontaneous or evoked activity of these cells. Noxious mechanical stimulation inhibited 59% of the cells receiving nociceptive inputs (wide dynamic range and nociceptive specific) but only 5% of the other cell types. Inhibition produced by mechanical stimulation lasted for the full duration of stimulus application (up to 30 s) whereas inhibition produced by electrical stimulation lasted less than 500 ms. Increasing the depth of anesthesia was found to depress or abolish the inhibition.     

Interactions between superficial and deep dorsal horn spinal cord neurons in the processing of nociceptive information

In acute rat spinal cord slices, the application of capsaicin (5 μm , 90 s), an agonist of transient receptor potential vanilloid 1 receptors expressed by a subset of nociceptors that project to laminae I–II of the spinal cord dorsal horn, induced an increase in the frequency of spontaneous excitatory and spontaneous inhibitory postsynaptic currents in about half of the neurons in laminae II, III–IV and V. In the presence of tetrodotoxin, which blocks action potential generation and polysynaptic transmission, capsaicin increased the frequency of miniature excitatory postsynaptic currents in only 30% of lamina II neurons and had no effect on the frequency of miniature excitatory postsynaptic currents in laminae III–V or on the frequency of miniature inhibitory postsynaptic currents in laminae II–V. When the communication between lamina V and more superficial laminae was interrupted by performing a mechanical section between laminae IV and V, capsaicin induced an increase in spontaneous excitatory postsynaptic current frequency in laminae II–IV and an increase in spontaneous inhibitory postsynaptic current frequency in lamina II that were similar to those observed in intact slices. However, in laminae III–IV of transected slices, the increase in spontaneous inhibitory postsynaptic current frequency was virtually abolished. Our results indicate that nociceptive information conveyed by transient receptor potential vanilloid 1‐expressing nociceptors is transmitted from lamina II to deeper laminae essentially by an excitatory pathway and that deep laminae exert a ‘feedback’ control over neurons in laminae III–IV by increasing inhibitory synaptic transmission in these laminae. Moreover, we provide evidence that laminae III–IV might play an important role in the processing of nociceptive information in the dorsal horn.     

Red nucleus modulation of somatosensory responses of cat spinal cord dorsal horn neurons

Single unit extracellular recordings were obtained from cat lumbar spinal cord dorsal horn neurons activated by cutaneous inputs. The effect of electrical conditioning stimuli applied to the red nucleus (RN) was predominantly that of inhibition although in some cases excitation was seen. All the neurons that projected to the lateral cervical nucleus were inhibited; none of these were excited. These findings suggest that the RN may exert a dynamic modulatory action on the transmission of cutaneous information during the execution of a motor program.     

Convergence of cutaneous, musculoskeletal, dural and visceral afferents onto nociceptive neurons in the first cervical dorsal horn

The convergence of cutaneous, musculoskeletal, dural and visceral afferents onto nociceptive neurons in the first cervical dorsal horn was investigated in urethane/chloralose-anesthetized rats. Electrical stimulation was applied to facial, neck, shoulder and forepaw skin, cornea (COR), dura, second cervical (C2) nerve, hypoglossal nerve, temporomandibular joint, masseter (MAS) muscle and superior laryngeal nerve. In addition, acetic acid was injected intraperitoneally and microinjection of glutamate was applied to the tongue, MAS muscle, splenius cervicis muscle, dura and intrapericardial area. A total of 52 nociceptive neurons classified as wide dynamic range (n = 28) or nociceptive-specific (n = 24) was studied. All nociceptive neurons received afferent input from the skin and at least one COR, musculoskeletal, dural or visceral afferent source in the trigeminal (V) or cervical area but input from afferent sources caudal to the C2 innervation territory was sparse. The proportion of neurons responding to COR, dural, C2 nerve, hypoglossal nerve, temporomandibular joint, MAS muscle and superior laryngeal nerve stimulations was 87, 54, 85, 52, 73, 64 and 31%, respectively. Electrical stimulation of all tested sites showed a double logarithmic stimulus-response relation, and cluster analysis of the excitability to COR, musculoskeletal, dural and visceral stimulations revealed two groups of neurons, one mainly containing wide dynamic range neurons and one mainly containing nociceptive-specific neurons. These findings indicate that afferent convergence in first cervical dorsal horn nociceptive neurons may be limited to the craniofacial area and that they may play an important role in the integration of craniofacial and upper cervical nociceptive inputs.     

Evidence for two populations of rat spinal dorsal horn neurons excited by urinary bladder distension

Spinal L6-S2 dorsal horn neurons of cervical spinal cord-transected, decerebrate female rats were characterized using urinary bladder distension (UBD) as a visceral stimulus. Constant pressure, phasic, graded (20-80 mm Hg, 20 s) air UBD was delivered via a transurethral catether and extracellular single-unit recordings obtained from all neurons excited by UBD. Responses to graded UBD and noxious/non-noxious cutaneous stimuli were determined in 258 neurons which could be stratified into two groups based on their effect of a counterirritation stimulus: Type I neurons (n=112) were inhibited by noxious pinch presented in a non-segmental field; Type II neurons (n=146) were not similarly inhibited. Both Types of neurons were identified in both superficial and deep recording sites and demonstrated graded responses to graded UBD. All UBD-excited neurons had convergent cutaneous receptive fields (RFs) excited by non-noxious and/or noxious stimuli. As a group, Type I neurons had a period of decreased activity following termination of the distending stimulus whereas Type II neurons typically had a sustained afterdischarge. UBD-evoked activity in Type II neurons was inhibited more than similar activity in Type I neurons by both intravenous morphine and lidocaine. These results support the assertion that at least two different populations of spinal dorsal horn neurons exist which encode for a stimulus of urinary bladder distension. These populations are an analogue to previously characterized, similar neuronal populations excited by colorectal distension and suggest that they are representative of the overall phenomenon of visceral sensory processing, a component of which is nociception.     

Nonsegmental inhibition of rat dorsal horn neurons by innocuous stimulation

In rats anesthetized with thiamylal sodium, responses of spinal cord dorsal horn neurons to noxious skin heating of the tail were recorded by extracellular microelectrodes. Inhibition of these responses by innocuous mechanical stimulation (light brushing) of the ipsilateral forelimb was assessed. Short-lasting application (3 min, or less) of light brushing did not inhibit neuronal responses to noxious heating. Long-lasting application (5 min, or more) inhibited responses of these neurons to noxious stimulation. The results indicate that, in the anesthetized rat, remotely applied innocuous cutaneous stimuli can inhibit nociceptive responses of dorsal horn neurons, if applied for a sufficiently long time.     

Quantitative study of NPY-expressing GABAergic neurons and axons in rat spinal dorsal horn

Between 25-40% of neurons in laminae I-III are GABAergic, and some of these express neuropeptide Y (NPY). We previously reported that NPY-immunoreactive axons form numerous synapses on lamina III projection neurons that possess the neurokinin 1 receptor (NK1r). The aims of this study were to determine the proportion of neurons and GABAergic boutons in this region that contain NPY, and to look for evidence that they selectively innervate different neuronal populations. We found that 4-6% of neurons in laminae I-III were NPY-immunoreactive and based on the proportions of neurons that are GABAergic, we estimate that NPY is expressed by 18% of inhibitory interneurons in laminae I-II and 9% of those in lamina III. GABAergic boutons were identified by the presence of the vesicular GABA transporter (VGAT) and NPY was found in 13-15% of VGAT-immunoreactive boutons in laminae I-II, and 5% of those in lamina III. For both the lamina III NK1r-immunoreactive projection neurons and protein kinase Cγ (PKCγ)-immunoreactive interneurons in lamina II, we found that around one-third of the VGAT boutons that contacted them were NPY-immunoreactive. However, based on differences in the sizes of these boutons and the strength of their NPY-immunoreactivity, we conclude that these originate from different populations of interneurons. Only 6% of VGAT boutons presynaptic to large lamina I projection neurons that lacked NK1rs contained NPY. These results show that NPY-containing neurons make up a considerable proportion of the inhibitory interneurons in laminae I-III, and that their axons preferentially target certain classes of dorsal horn neuron.     

Optical responses evoked by single-pulse stimulation to the dorsal root in the rat spinal dorsal horn in slice

Neuronal excitation evoked after dorsal-root (DR) stimulation in the spinal dorsal horn (DH) of rats was visualized with a high-resolution optical-imaging method, and the propagation mechanism was studied. Transverse slices of the spinal cord were obtained from 2–4 week-old rats and stained with the voltage-sensitive dye RH-482. Single-pulse stimulation to the primary-afferent A fibers in the DR attached to the slice evoked a weak, brief (<10 ms) excitatory optical response in the laminae I and III–V. When the stimulus intensity and duration were increased to activate both A and C fibers, an additional, much greater, and longer-lasting (>100 ms) excitatory response was generated in the laminae I–III, most intensely in the lamina II. A treatment with excitatory amino acid (EAA) antagonists, -2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione, significantly reduced the amplitude and duration of the response in the lamina II. The optical response in the antagonists-containing solution was quite similar to that recorded in a Ca²⁺-free solution that blocked afferent synaptic transmission. The late component (>10 ms) was, however, slightly greater than that in the Ca²⁺-free solution. Treatment with the ATP-receptor antagonist, suramin, had a minimal effect on the response in the presence of EAA antagonists. These results suggested that the propagation of the DR-stimulus-elicited excitation was contributed largely by EAA receptors, but also by other receptors to a much lesser extent.     

Sodium salicylate reduces gamma aminobutyric acid-induced current in rat spinal dorsal horn neurons

Sodium salicylate is one of the nonsteroidal antiinflammatory drugs and is clinically used for antiinflammation and chronic pain relief. In the present study, we investigated the actions of sodium salicylate on gamma-aminobutyric acid type A receptor (GABA(A)) current in cultured rat spinal dorsal horn neurons. Sodium salicylate was found to reduce GABA(A) current in a reversible and concentration-dependent manner, but did not change its ion selectivity. Sodium salicylate was effective only when GABA and sodium salicylate were applied together. Application of sodium salicylate immediately before, but not during, the application of GABA did not result in a significant reduction of GABA(A) current. Our results demonstrate that sodium salicylate reversibly attenuates the GABA(A) response of dorsal horn neurons, suggesting that GABA(A) receptors in the region are pharmacological targets of sodium salicylate.     

Electrical stimulation of thalamic Nucleus Submedius inhibits responses of spinal dorsal horn neurons to colorectal distension in the rat

In 78 halothane-anesthetized rats, we characterized the responses of single neurons in the dorsal horn of L(6)-S(1) spinal segments to a noxious visceral stimulus (colorectal balloon distension, CRD), and studied the effects of focal electrical stimulation of Nucleus Submedius (Sm) on these responses using standard extracellular microelectrode recording techniques. A total of 102 neurons were isolated on the basis of spontaneous activity. Eighty (78%) responded to CRD, of which 70% had excitatory and 30% had inhibitory responses. Neurons showed graded responses to graded CRD pressures (20-100 mmHg), with maximum excitation or inhibition occurring at 100 mmHg. Responses to noxious (pinch, heat) and innocuous (brush, tap) cutaneous stimuli were studied in 73 of the spinal dorsal horn neurons isolated. Fifty-seven (78%) of these neurons (46 CRD-responsive and 11 CRD-nonresponsive) had cutaneous receptive fields, of which 35 (61%) were small and ipsilateral, 14 (25%) were large and ipsilateral, 7 (12%) were large or small and bilateral, and 1 (2%) was small and contralateral. Sixty-one percent of these neurons responded to both noxious and innocuous cutaneous stimulation, 35% responded only to noxious stimulation, and 4% responded only to innocuous stimulation. Electrical stimulation (50-300 microA) of the contralateral Sm produced intensity-dependent attenuation of the CRD-evoked activities of most neurons (18/28 of CRD-excited and 7/12 of CRD-inhibited) tested. Sm stimulation produced facilitation of CRD responses of only one neuron (CRD-inhibited). Sm stimulation had no effects on spontaneous activity. These data indicate that Sm may be involved in the descending inhibitory modulation of visceral nociception at the spinal level.     

Regulation by equilibrative nucleoside transporter of adenosine outward currents in adult rat spinal dorsal horn neurons

A current response induced by superfusing adenosine was examined in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. In 78% of the neurons examined, adenosine induced an outward current at -70 mV [18.8 +/- 1.1 pA (n = 98) at 1mM] in a dose-dependent manner (EC(50) = 177 microM). A similar current was induced by A(1) agonist N(6)-cyclopentyladenosine (1 microM), whereas A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 microM) reversed the adenosine action. The adenosine current reversed its polarity at a potential being close to the equilibrium potential for K(+), and was attenuated by Ba(2+) (100 microM) and 4-aminopyridine (5mM) but not tetraethylammonium (5mM). The adenosine current was enhanced in duration by equilibrative nucleoside-transport (rENT1) inhibitor S-(4-nitrobenzyl)-6-thioinosine (1 microM) and adenosine deaminase (ADA) inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (1 microM), and slowed in falling phase by adenosine kinase (AK) inhibitor iodotubercidine (1 microM). We conclude that a Ba(2+)- and 4-aminopyridine-sensitive K(+) channel in SG neurons is opened via the activation of A(1) receptors by adenosine whose level is possibly regulated by rENT1, adenosine deaminase and adenosine kinase. Considering that intrathecally-administered adenosine analogues produce antinociception, the regulatory systems of adenosine may serve as targets for antinociceptive drugs.     

Role of substance P in the modulation of C-fiber-evoked responses of spinal dorsal horn neurons

Substance P (SP) as well as excitatory amino acids (EAAs) appear to be released in response to stimulation of primary afferent C-fibers. Activity atN-methyl-d-aspartate (NMDA) receptors is essential for wind-up (the progressive potentiation of C-fiber-evoked responses of single neurons in response to an electrical stimulation), however, the role of SP in wind-up is unclear. To address this, the effects of iontophoretically applied CP-99,994 (a NK-1 receptor antagonist), SP and SP(1–7) (an N-terminal breakdown product of SP), were compared on responses of spinal dorsal horn wide dynamic range (WDR) neurons of the rat. Post-stimulus time histograms (PSTH) were summed over 12 responses to low frequency (0.5 Hz) electrical stimulation of the cutaneous receptive field. Changes in responses of dorsal horn neurons were evaluated by monitoring C-fiber input, wind-up, and the total number of spikes evoked by C-fiber activity in response to the 12 stimuli. The NK-1 receptor antagonist CP-99,994 significantly inhibited the total number of C-spikes and caused a significant reduction in wind-up without changing the C-fiber input, indicating the involvement of NK-1 receptors in wind-up. Application of SP led to an overall increase in the total number of C-fiber evoked responses of dorsal horn neurons and ('-fiber input, however, wind-up, as defined, was significantly decreased following SP. In contrast, substance P(1–7) evoked a long-lasting increase in the total number of C-fiber-related spikes which was initially sustained by a long-lasting increase in the input followed by a longer lasting increase in wind-up, an effect opposite that of CP-99,994. As NMDA activity has been previously shown to be inhibited and then potentiated by SP N-terminal activity over a similar time interval, the present data are consistent with the mediation of wind-up by NMDA and its modulation by SP N-terminal activity. Release of SP in response to noxious stimulation may, therefore, increase primary afferent C-fiber activity (input) whereas an accumulation of SP N-terminal metabolites appears to potentiate wind-up, perhaps via positive modulation of EAA activity.