Sensory Outflow Research Articles

Afferent Nerve Activity Ex Vivo and Voiding Frequency In Vivo are Driven by Bladder Filling Rate in a Mouse Model of Polydipsia‐Polyuria

The mechanisms regulating urinary voiding function are not entirely understood. Many disorders of the lower urinary tract such as overactive bladder, urinary incontinence, and underactive bladder are associated with dysfunctional voiding behavior. Developing treatments to improve voiding function in afflicted individuals has proved to be problematic, in part due to a lack of basic understanding of the mechanisms responsible for controlling voiding function.We have shown previously in an ex vivo bladder model that afferent nerve activity increases as the bladder is filled. Furthermore, transient pressure events occurring during bladder filling trigger substantial rises in afferent nerve activity, more so than would occur during a similar rise in bladder pressure associated with bladder filling. This observation suggests that localized wall deformations associated with transient pressure events could transduce important sensory information to the central nervous system as to the filling state of the bladder. In the present study, we hypothesized that bladder filling rate is an important parameter that controls voiding function in vivo. We used an acute sucrose induced polydipsia/polyuria model that allows us to determine the impact of bladder filling rate on voiding behavior noninvasively in conscious freely moving mice.Male C57Bl6 mice (6 weeks old) were placed into a UroVoid apparatus for 48 hour voiding studies. One group of mice had access to tap water while a second group of mice had access to 5% sucrose in water. All mice had access to standard rodent chow. The UroVoid apparatus allows us to quantify the time and volume for each micturition.Mice drinking 5% sucrose consumed substantially more fluid than mice consuming tap water (26.98 ± 13.12 ml vs. 7.23 ± 1.35 ml). This polydipsia was reflected in a profound polyuria in mice consuming sucrose, as these mice voided a total of 18.98 ± 13.52 ml of urine over 79 ± 42 voids compared to 3.91 ± 0.54 ml of urine over 22 ± 16 voids in mice consuming tap water. Using the voided urine volume and the intermicturition interval for each void, we calculated that the urine production rate (UPR) is ~2 μl/min in mice consuming tap water, whereas the UPR is greatly elevated to ~25 μl/min in mice consuming 5% sucrose. Voiding frequency is greatly increased in mice consuming 5% sucrose, which we believe reflects underlying sensory function.To address the impact of bladder filling rate on sensory function, we measured afferent pelvic nerve activity in ex vivo bladder preparations at filling rates of 3.3, 10, and 30 μl/min. As expected, sensory nerve activity was increased at higher filling rates. For example, at an intravesical pressure of 15–20 mmHg, afferent nerve frequency increased from 91.5 ± 43.0 Hz at 3.3 μl/min to 174.9 ± 64.1 Hz at 30 μl/min.In conclusion, sucrose‐induced polydipsia/polyuria in mice is an effective way to examine the impact of bladder filling rate on voiding behavior. Increases in bladder filling rate are associated with increases in afferent nerve outflow in the bladder, and this increase in sensory outflow may contribute to elevated voiding frequency at higher bladder filling rates.Support or Funding InformationSupported by NIH R37 DK053832

Smooth Muscle Tone Contributes to Bladder Wall Stiffness during Filling

Sensations of bladder fullness are conveyed to the central nervous system by sensory nerves as a response to distention of the urinary bladder. This distention is accompanied by non‐voiding transient pressure events and contractions called “micromotions” that are responsible for the majority of sensory outflow during bladder filling. Changes in smooth muscle contractility along with alterations in wall stiffness and sensory feedback are observed in most UB pathologies. Thus, the mechanical properties of bladder wall may play a crucial role in communicating a sense of bladder fullness. We hypothesize that the contractile apparatus of the urinary bladder is actively involved in maintaining bladder wall stiffness during filling.Whole mouse bladders were dissected and mounted in a custom designed pentaplanar reflected image macroscopy (PRIM) system, for simultaneous measurement of bladder wall micromotions, bladder volume, intravesical pressure, bladder wall stress, and bladder wall strain during ex vivo bladder filling. Bladders were filled at a constant rate (30 μl/min) until intravesical pressure reached 25 mmHg. During filling, bladder wall micromotions led to transient pressure events (0–2 mmHg in amplitude). These micromotions began on the lateral sides of the bladder wall near the trigone and expanded upwards as the bladder filled. Baseline bladder pressure, however, remained relatively low over a large infused volume (~350 μL). Next, we derived the stress versus strain relationship of the bladder wall from the pressure‐volume recordings during filling. At baseline pressures >15 mmHg, intravesical pressure rapidly increased and transient pressure events could no longer be distinguished implying that wall compliance is maintained until larger infused volumes, after which bladder wall stress with respect to strain increases rapidly. To determine if smooth muscle tone altered bladder wall stress during filling, bladder was exposed to myosin inhibitor blebbistatin (10 μM) or PI3 kinase inhibitor wortmannin (10 μM). Both blebbistatin and wortmannin effectively diminished the micromotions and shifted the stress vs strain curve to the right, indicating that the smooth muscle contractile apparatus and smooth muscle tone contribute to bladder wall stiffness, but predominantly only at higher filling volumes. These findings suggest that smooth muscle tone directly increases bladder wall stiffness and that changes in this tone could alter sensory feedback during bladder filling.Support or Funding InformationSupported by NIH K01DK103840 (NRT) and R01DK119615 (NRT).

The KV 7 channel activator retigabine suppresses mouse urinary bladder afferent nerve activity without affecting detrusor smooth muscle K+ channel currents.

KV 7 channels are a family of voltage-dependent K+ channels expressed in many cell types, which open in response to membrane depolarization to regulate cell excitability. Drugs that target KV 7 channels are used clinically to treat epilepsy. Interestingly, these drugs also cause urinary retention, but it was unclear how. In this study, we focused on two possible mechanisms by which retigabine could cause urinary retention: by decreasing smooth muscle excitability, or by decreasing sensory nerve outflow. Urinary bladder smooth muscle had no measurable KV 7 channel currents. However, the KV 7 channel agonist retigabine nearly abolished sensory nerve outflow from the urinary bladder during bladder filling. We conclude that KV 7 channel activation likely affects urinary bladder function by blocking afferent nerve outflow to the brain, which is key to sensing bladder fullness. KV 7 channels are voltage-dependent K+ channels that open in response to membrane depolarization to regulate cell excitability. KV 7 activators, such as retigabine, were used to treat epilepsy but caused urinary retention. Using electrophysiological recordings from freshly isolated mouse urinary bladder smooth muscle (UBSM) cells, isometric contractility of bladder strips, and ex vivo measurements of bladder afferent activity, we explored the role of KV 7 channels as regulators of murine urinary bladder function. The KV 7 activator retigabine (10μM) had no effect on voltage-dependent K+ currents or resting membrane potential of UBSM cells, suggesting that these cells lacked retigabine-sensitive KV 7 channels. The KV 7 inhibitor XE-991 (10μM) inhibited UBSM K+ currents; the properties of these currents, however, were typical of KV 2 channels and not KV 7 channels. Retigabine inhibited voltage-dependent Ca2+ channel (VDCC) currents and reduced steady-state contractions to 60mM KCl in bladder strips, suggesting that reduction in VDCC current was sufficient to directly affect UBSM function. To determine if retigabine altered ex vivo bladder sensory outflow, we measured afferent activity during simulated transient contractions (TCs) of the bladder wall. Simulated TCs caused bursts of afferent activity that were nearly abolished by retigabine. The effects of retigabine were blocked by co-incubation with XE-991, suggesting specific activation of KV 7 channels on afferent nerves. These results indicate that retigabine primarily affects urinary bladder function by inhibiting TC generation and afferent nerve activity, which are key to sensing bladder fullness. Any direct inhibition of UBSM contractility is likely to be from non-specific effects on VDCCs and KV 2 channels.

Development of stress-induced bladder insufficiency requires functional TRPV1 channels.

Social stress causes profound urinary bladder dysfunction in children that often continues into adulthood. We previously discovered that the intensity and duration of social stress influences whether bladder dysfunction presents as overactivity or underactivity. The transient receptor potential vanilloid type 1 (TRPV1) channel is integral in causing stress-induced bladder overactivity by increasing bladder sensory outflow, but little is known about the development of stress-induced bladder underactivity. We sought to determine if TRPV1 channels are involved in bladder underactivity caused by stress. Voiding function, sensory nerve activity, and bladder wall remodeling were assessed in C57BL/6 and TRPV1 knockout mice exposed to intensified social stress using conscious cystometry, ex vivo afferent nerve recordings, and histology. Intensified social stress increased void volume, intermicturition interval, bladder volume, and bladder wall collagen content in C57BL/6 mice, indicative of bladder wall remodeling and underactive bladder. However, afferent nerve activity was unchanged and unaffected by the TRPV1 antagonist capsazepine. Interestingly, all indices of bladder function were unchanged in TRPV1 knockout mice in response to social stress, even though corticotrophin-releasing hormone expression in Barrington's Nucleus still increased. These results suggest that TRPV1 channels in the periphery are a linchpin in the development of stress-induced bladder dysfunction, both with regard to increased sensory outflow that leads to overactive bladder and bladder wall decompensation that leads to underactive bladder. TRPV1 channels represent an intriguing target to prevent the development of stress-induced bladder dysfunction in children.

Pharmacological analysis of the inhibition produced by moxonidine and agmatine on the vasodepressor sensory CGRPergic outflow in pithed rats

Calcitonin gene-related peptide (CGRP) plays a role in several (patho)physiological functions, and modulation of its release is considered a therapeutic target. In this respect, electrical spinal (T9­-T12) stimulation of the perivascular sensory outflow in pithed rats produces vasodepressor responses mediated by CGRP release. This study investigated the role of imidazoline I1 and I2 receptors in the inhibition by moxonidine and agmatine of these vasodepressor responses. Male Wistar pithed rats (pretreated i.v. with 25mg/kg gallamine and 2mg/kg⋅min hexamethonium) received i.v. continuous infusions of methoxamine (20μg/kg⋅min) followed by physiological saline (0.02ml/min), moxonidine (1, 3, 10 or 30μg/kg⋅min) or agmatine (1000 or 3000μg/kg⋅min). Under these conditions, electrical stimulation (0.56–5.6Hz; 50V; 2ms) of the spinal cord (T9-T12) produced frequency-dependent vasodepressor responses which were: (i) unchanged during saline infusion; and (ii) inhibited during the above infusions of moxonidine or agmatine. Moreover, using i.v. administrations, the inhibition by 3μg/kg⋅min moxonidine or 3000μg/kg⋅min agmatine (which failed to inhibit the vasodepressor responses by α-CGRP; 0.1–1µg/kg) was: (i) unaltered after saline (1ml/kg), rauwolscine (300μg/kg; α2-adrenoceptor antagonist) or BU224 (300μg/kg; imidazoline I2 receptor antagonist); and (ii) reversed after AGN 192403 (3000μg/kg; imidazoline I1 receptor antagonist). This reversion was relatively more pronounced after AGN 192403 plus rauwolscine. These blocking doses of antagonists lacked any effects on the electrically-induced vasodepressor responses. Therefore, the inhibition of the vasodepressor sensory CGRPergic outflow by moxonidine and agmatine is mainly mediated by prejunctional imidazoline I1 receptors on perivascular sensory nerves.

Transient contractions of urinary bladder smooth muscle are drivers of afferent nerve activity during filling.

Activation of afferent nerves during urinary bladder (UB) filling conveys the sensation of UB fullness to the central nervous system (CNS). Although this sensory outflow is presumed to reflect graded increases in pressure associated with filling, UBs also exhibit nonvoiding, transient contractions (TCs) that cause small, rapid increases in intravesical pressure. Here, using an ex vivo mouse bladder preparation, we explored the relative contributions of filling pressure and TC-induced pressure transients to sensory nerve stimulation. Continuous UB filling caused an increase in afferent nerve activity composed of a graded increase in baseline activity and activity associated with increases in intravesical pressure produced by TCs. For each ∼4-mmHg pressure increase, filling pressure increased baseline afferent activity by ∼60 action potentials per second. In contrast, a similar pressure elevation induced by a TC evoked an ∼10-fold greater increase in afferent activity. Filling pressure did not affect TC frequency but did increase the TC rate of rise, reflecting a change in the length-tension relationship of detrusor smooth muscle. The frequency of afferent bursts depended on the TC rate of rise and peaked before maximum pressure. Inhibition of small- and large-conductance Ca(2+)-activated K(+) (SK and BK) channels increased TC amplitude and afferent nerve activity. After inhibiting detrusor muscle contractility, simulating the waveform of a TC by gently compressing the bladder evoked similar increases in afferent activity. Notably, afferent activity elicited by simulated TCs was augmented by SK channel inhibition. Our results show that afferent nerve activity evoked by TCs represents the majority of afferent outflow conveyed to the CNS during UB filling and suggest that the maximum TC rate of rise corresponds to an optimal length-tension relationship for efficient UB contraction. Furthermore, our findings implicate SK channels in controlling the gain of sensory outflow independent of UB contractility.

Pharmacological evidence that histamine H3 receptors inhibit the vasodepressor responses by selective stimulation of the rat perivascular sensory CGRPergic outflow

This study has investigated whether pharmacological activation of Gi/o coupled histamine H3/H4 receptors inhibits the rat vasodepressor sensory outflow. For this purpose, 100 male Wistar rats were pithed, artificially ventilated and pretreated (i.v.) with: 25mg/kg gallamine, 2mg/kg/min hexamethonium and 20μg/kg/min methoxamine, followed by i.v. continuous infusions of physiological saline (0.02ml/min) or immepip (3.1, 10 or 31μg/kg/min; a histamine H3/H4 receptor agonist). Under these conditions, electrical stimulation (0.56–5.6Hz; 50V and 2ms) of the spinal cord (T9–T12) resulted in frequency-dependent vasodepressor responses, which were: (i) unchanged during the infusions of saline or immepip (3.1μg/kg/min); and (ii) significantly but, surprisingly, not dose-dependently inhibited by 10 and 31μg/kg/min immepip. Moreover, the sensory-inhibition by 10μg/kg/min immepip (which failed to inhibit the vasodepressor responses by i.v. bolus injections of α-CGRP; 0.1–1µg/kg) was: (i) essentially unaltered after i.v. administration of saline (1ml/kg) or blocking doses of the antagonists ketotifen (100μg/kg; H1), ranitidine (1000μg/kg; H2) or JNJ7777120 (310μg/kg; H4); and (ii) abolished after i.v. thioperamide (310µg/kg; H3). In conclusion, our results suggest that immepip-induced inhibition of the vasodepressor sensory outflow is mainly mediated by prejunctional activation of histamine H3 receptors.

Role of 5-HT7 receptors in the inhibition of the vasodepressor sensory CGRPergic outflow in pithed rats

The role of calcitonin gene-related peptide (CGRP) in the modulation of vascular tone has been widely documented. Indeed, electrical stimulation of the perivascular sensory outflow in pithed rats induces vasodepressor responses by activation of CGRP receptors. This study investigated the role of 5-HT7 receptors in the inhibition of the rat vasodepressor sensory outflow. Male Wistar pithed rats were pretreated with i.v. continuous infusions of hexamethonium and methoxamine, followed by physiological saline or AS-19 (a 5-HT7 receptor agonist). Then, electrical stimulation of the spinal cord resulted in frequency-dependent decreases in DBP. The infusions of AS-19, as compared to those of saline, inhibited the vasodepressor responses induced by electrical stimulation without affecting those to i.v. bolus injections of exogenous α-CGRP. This inhibition by AS-19 was abolished by the antagonists pimozide (5-HT7) or sulfisoxazole (ETA), but not by indomethacin (COX1/2) or losartan (AT1), at doses that did not affect per se the electrically-induced vasodepressor responses. Interestingly, glibenclamide (an ATP-dependent K+ channel blocker) attenuated these vasodepressor responses. The present results suggest that AS-19-induced inhibition of the rat vasodepressor sensory CGRPergic outflow is mainly mediated by 5-HT7 receptors via endothelin release, with the possible involvement of ATP-dependent K+ channels.

Role of Pre‐Junctional CB1, But not CB2, TRPV1 or GPR55 Receptors in Anandamide‐Induced Inhibition of the Vasodepressor Sensory CGRPergic Outflow in Pithed Rats

Stimulation of the perivascular sensory outflow in pithed rats produces vasodepressor responses mediated by CGRP release. Interestingly, endocannabinoids such as anandamide (which interacts with CB1 , CB2 , TRPV1 and GPR55 receptors) can regulate the activity of perivascular sensory nerves in dural blood vessels by modulating CGRP release. Yet, as no publication has reported whether this mechanism is operative in the healthy systemic vasculature, this study has specifically analysed the receptors mediating the potential inhibitory effects of the cannabinoid (CB) receptor agonists anandamide (non-selective), JWH-015 (CB2 ) and lysophosphatidylinositol (GPR55) on the rat vasodepressor sensory CGRPergic outflow (an index of systemic vasodilatation). Healthy pithed rats were pre-treated with consecutive i.v. continuous infusions of hexamethonium, methoxamine and the above agonists. Electrical spinal (T9 -T12 ) stimulation of the vasodepressor sensory CGRPergic outflow or i.v. injections of α-CGRP produced frequency-dependent or dose-dependent vasodepressor responses. The infusions of anandamide in a dose-dependent manner inhibited the vasodepressor responses by electrical stimulation (remaining unaffected by JWH-015 or lysophosphatidylinositol), but not those by α-CGRP. After i.v. administration of antagonists, the inhibition by 3.1 μg/kg min anandamide was: (i) potently blocked by 31-100 μg/kg NIDA41020 (CB1 ), (ii) unaffected by 180 μg/kg AM630 (CB2 ), 31 μg/kg cannabidiol (GPR55) or 31-100 μg/kg capsazepine (TRPV1) and (iii) slightly blocked by 310 μg/kg AM630. The above doses of antagonists were enough to block their respective receptors. These results suggest that anandamide-induced inhibition of the vasodepressor sensory CGRPergic outflow is mainly mediated by pre-junctional activation of CB1 receptors, with no pharmacological evidence for the role of CB2 , TRPV1 or GPR55 receptors.

The Role of Pre-junctional D2-like Receptors Mediating Quinpirole-Induced Inhibition of the Vasodepressor Sensory CGRPergic Out-flow in Pithed Rats

Calcitonin gene-related peptide (CGRP) released from perivascular sensory nerves plays a role in the regulation of vascular tone. Indeed, electrical stimulation of the perivascular sensory out-flow in pithed rats produces vasodepressor responses, which are mainly mediated by CGRP release. This study investigated the potential role of dopamine D1 -like and D2 -like receptors in the inhibition of these vasodepressor responses. For this purpose, male Wistar pithed rats (pre-treated i.v. with 25 mg/kg gallamine and 2 mg/kg min. hexamethonium) received i.v. continuous infusions of methoxamine (20 μg/kg min.) followed by physiological saline (0.02 ml/min.), the D1 -like receptor agonist SKF-38393 (0.1-1 μg/kg min.) or the D2 -like receptor agonist quinpirole (0.03-10 μg/kg min.). Under these conditions, electrical stimulation (0.56-5.6 Hz; 50 V and 2 ms) of the thoracic spinal cord (T9 -T12 ) resulted in frequency-dependent vasodepressor responses which were (i) unchanged during the infusions of saline or SKF-38393 and (ii) inhibited during the infusions of quinpirole (except at 0.03 μg/kg min.). Moreover, the inhibition induced by 0.1 μg/kg min. quinpirole (which failed to inhibit the vasodepressor responses elicited by i.v. bolus injections of exogenous α-CGRP; 0.1-1 μg/kg) was (i) unaltered after i.v. treatment with 1 ml/kg of either saline or 5% ascorbic acid and (ii) abolished after 300 μg/kg (i.v.) of the D2 -like receptor antagonists haloperidol or raclopride. These doses of antagonists (enough to completely block D2 -like receptors) essentially failed to modify per se the electrically induced vasodepressor responses. In conclusion, our results suggest that quinpirole-induced inhibition of the vasodepressor sensory CGRPergic out-flow is mainly mediated by pre-junctional D2 -like receptors.

The 5-HT 1 receptors inhibiting the rat vasodepressor sensory CGRPergic outflow: Further involvement of 5-HT 1F, but not 5-HT 1A or 5-HT 1D, subtypes

We have previously shown that 5-HT 1B receptors inhibit prejunctionally the rat vasodepressor CGRPergic sensory outflow. Since 5-HT 1 receptors comprise 5-HT 1A, 5-HT 1B, 5-HT 1D and 5-HT 1F functional subtypes, this study has further investigated the role of 5-HT 1A, 5-HT 1D and 5-HT 1F receptor subtypes in the inhibition of the above vasodepressor sensory outflow. Pithed rats were pretreated with i.v. continuous infusions of hexamethonium and methoxamine, followed by 5-HT 1 receptor agonists. Then electrical spinal stimulation (T 9–T 12) or i.v. bolus injections of exogenous α-CGRP produced frequency-dependent or dose-dependent vasodepressor responses. The electrically-induced vasodepressor responses remained unchanged during infusions of the 5-HT 1A receptor agonists 8-OH-DPAT and NN-DP-5-CT. In contrast, these responses were inhibited by the agonists sumatriptan (5-HT 1A/1B/1D/1F), indorenate (5-HT 1A), PNU-142633 (5-HT 1D) or LY344864 (5-HT 1F), which did not affect the vasodepressor responses to exogenous CGRP (implying a prejunctional sensory-inhibition). When analysing the effects of antagonists: (i) 310 μg/kg (but not 100 μg/kg) GR127935 (5-HT 1A/1B/1D/1F) abolished the inhibition to sumatriptan, indorenate, PNU-142633 or LY344864; (ii) 310 μg/kg SB224289 (5-HT 1B) or BRL15572 (5-HT 1D) failed to block the inhibition to sumatriptan or PNU-142633, whereas SB224289 + BRL15572 partly blocked the inhibition to sumatriptan; and (iii) 10 μg/kg WAY100635 (5-HT 1A) failed to block the inhibition to indorenate. These results suggest that 5-HT 1F, but not 5-HT 1A or 5-HT 1D, receptor subtypes inhibit the vasodepressor sensory CGRPergic outflow although, admittedly, no selective 5-HT 1F receptor agonist is available yet. The pharmacological profile of these receptors resembles that shown in rat dorsal root ganglia by molecular biology techniques.

Activation of 5-HT1B receptors inhibits the vasodepressor sensory CGRPergic outflow in pithed rats

The importance of calcitonin gene-related peptide (CGRP) in the regulation of vascular tone has been widely documented. Indeed, stimulation of the perivascular sensory outflow in pithed rats results in vasodepressor responses, which are mediated by CGRP release. These vasodepressor responses are inhibited by clonidine via prejunctional alpha(2A/2C)-adrenoceptors, but no study has yet reported the role of prejunctional 5-hydroxytryptamine (5-HT) receptors in this experimental model. Since activation of prejunctional 5-HT(1) receptors results in inhibition of neurotransmitter release, this study sets out to investigate as an initial approach the role of 5-HT(1B) receptors in the inhibition of the vasodepressor sensory outflow in pithed rats. Male Wistar pithed rats were pretreated with hexamethonium (2mg/kg.min) followed by i.v. continuous infusions of methoxamine (20 microg/kg min), and then by saline (0.02 ml/min) or CP-93,129 (a rodent 5-HT(1B) receptor agonist; 0.1, 1 and 10 microg/kg min). Under these conditions, electrical stimulation (0.56-5.6 Hz; 50 V and 2 ms) of the spinal cord (T(9)-T(12)) resulted in frequency-dependent decreases in diastolic blood pressure. The infusions of CP-93,129, as compared to those of saline, inhibited the vasodepressor responses induced by electrical stimulation without affecting those to i.v. bolus injections of exogenous alpha-CGRP (0.1, 0.18, 0.31, 0.56 and 1 microg/kg). This inhibition by CP-93,129 was abolished by the antagonists GR127935 (5-HT(1B/1D)) or SB224289 (5-HT(1B)), but not by BRL15572 (5-HT(1D)). The above results suggest that CP-93,129-induced inhibition of the vasodepressor (perivascular) sensory outflow in pithed rats is mainly mediated by activation of prejunctional 5-HT(1B) receptors.

Effect of some acute and prophylactic antimigraine drugs on the vasodepressor sensory CGRPergic outflow in pithed rats

Aims This study analyzed in pithed rats the effect of several acute and prophylactic antimigraine drugs on the CGRPergic vasodepressor sensory outflow, in an attempt to investigate systemic cardiovascular effects in a model unrelated to migraine. Main methods Male Wistar pithed rats were pretreated with continuous i.v. infusions of hexamethonium (2 μg/kg.min; to block autonomic outflow) and methoxamine (15–20 μg/kg.min; to maintain diastolic blood pressure at around 130 mmHg). Under these conditions, the effect of both electrical stimulation (0.56–5.6 Hz; 50 V and 2 ms) of the spinal cord (T 9–T 12) or i.v. bolus injections of exogenous α-CGRP (0.1–1 µg/kg) were studied in animals pretreated with continuous i.v. infusions of sumatriptan (1–100 μg/kg.min), ergotamine (0.18–0.56 μg/kg.min), dihydroergotamine (1–10 μg/kg.min), magnesium valproate (1000–1800 μg/kg.min), propranolol (100–300 μg/kg.min) or their respective vehicles. Key findings Electrical stimulation of the spinal cord and i.v. bolus injections of exogenous α-CGRP resulted in, respectively, frequency- and dose-dependent decreases in diastolic blood pressure without affecting heart rate. Moreover, the infusions of sumatriptan, ergotamine and dihydroergotamine, but not of magnesium valproate, propranolol or their respective vehicles, dose-dependently inhibited the vasodepressor responses to electrical stimulation. In contrast, sumatriptan (10 μg/kg.min), ergotamine (0.31 μg/kg.min) and dihydroergotamine (3 μg/kg.min) failed to inhibit the vasodepressor responses to exogenous α-CGRP. Significance The above findings suggest that the acute (rather than the prophylactic) antimigraine drugs attenuate the vasodepressor sensory outflow mainly by prejunctional mechanisms. This may be of particular relevance when considering potential cardiovascular adverse effects by acute antimigraine drugs.

Pharmacological profile of the clonidine‐induced inhibition of vasodepressor sensory outflow in pithed rats: correlation with a2A/2C‐adrenoceptors

British Journal of Pharmacology (2008) 154, 1160; doi:10.1038/bjp.2008.250

Pharmacological profile of the clonidine‐induced inhibition of vasodepressor sensory outflow in pithed rats: correlation with α2A/2C‐adrenoceptors

Resistance blood vessels are innervated by sympathetic and primary sensory nerves, which modulate vascular tone through the release of noradrenaline and calcitonin gene-related peptide (CGRP), respectively. Moreover, electrical stimulation of the perivascular sensory outflow in pithed rats results in vasodepressor responses which are mainly mediated by CGRP release. The present study has investigated the role of alpha(2)-adrenoceptors in the inhibition of these vasodepressor responses. 144 pithed male Wistar rats were pretreated with hexamethonium (2 mg kg(-1) min(-1)) followed by i.v. continuous infusions of either methoxamine (15 and 30 microg kg(-1) min(-1)) or clonidine (3, 10 and 30 microg kg(-1) min(-1)). Under these conditions, electrical stimulation (0.56-5.6 Hz; 50 V and 2 ms) of the spinal cord (T(9)-T(12)) resulted in frequency-dependent decreases in diastolic blood pressure. The infusion of clonidine (10 microg kg(-1) min(-1)), as compared to those of methoxamine (15 or 30 microg kg(-1) min(-1)), inhibited the vasodepressor responses to electrical stimulation without affecting those to i.v. bolus injections of alpha-CGRP (0.1-1 microg kg(-1)). This inhibition by clonidine was: (i) antagonized by 300 microg kg(-1) rauwolscine (alpha(2A/2B/2C)), 300 and 1000 microg kg(-1) BRL44408 (alpha(2A)), or 10 and 30 microg kg(-1) MK912 (alpha(2C)); and (ii) unaffected by 1 ml kg(-1) saline, 100 microg kg(-1) BRL44408, 3000 and 10,000 microg kg(-1) imiloxan (alpha(2B)) or 3 microg kg(-1) MK912. The inhibition produced by 10 microg kg(-1) min(-1) clonidine on the vasodepressor (perivascular) sensory outflow in rats may be mainly mediated by prejunctional alpha(2A)/alpha(2C)-adrenoceptors.

P.1.d.018 Effect of dopamine depletion and 5-HT1B receptors blockade on the behavioural effect of various antidepressant in the mice FST

The importance of calcitonin gene-related peptide (CGRP) in the regulation of vascular tone has been widely documented. Indeed, stimulation of the perivascular sensory outflow in pithed rats results in vasodepressor responses, which are mediated by CGRP release. These vasodepressor responses are inhibited by clonidine via prejunctional α2A/2C-adrenoceptors, but no study has yet reported the role of prejunctional 5-hydroxytryptamine (5-HT) receptors in this experimental model. Since activation of prejunctional 5-HT1 receptors results in inhibition of neurotransmitter release, this study sets out to investigate as an initial approach the role of 5-HT1B receptors in the inhibition of the vasodepressor sensory outflow in pithed rats. Male Wistar pithed rats were pretreated with hexamethonium (2 mg/kg.min) followed by i.v. continuous infusions of methoxamine (20 µg/kg.min), and then by saline (0.02 ml/min) or CP-93,129 (a rodent 5-HT1B receptor agonist; 0.1, 1 and 10 µg/kg.min). Under these conditions, electrical stimulation (0.56-5.6 Hz; 50 V and 2 ms) of the spinal cord (T9-T12) resulted in frequency-dependent decreases in diastolic blood pressure. The infusions of CP-93,129, as compared to those of saline, inhibited the vasodepressor responses induced by electrical stimulation without affecting those to i.v. bolus injections of exogenous α-CGRP (0.1, 0.18, 0.31, 0.56 and 1 μg/kg). This inhibition by CP-93,129 was abolished by the antagonists GR127935 (5-HT1B/1D) or SB224289 (5-HT1B), but not by BRL15572 (5-HT1D). The above results suggest that CP-93,129-induced inhibition of the vasodepressor (perivascular) sensory outflow in pithed rats is mainly mediated by activation of prejunctional 5-HT1B receptors.

Spontaneous Discharge and Peripherally Evoked Orofacial Responses of Trigemino–Thalamic Tract Neurons during Wakefulness and Sleep

In the present study, ongoing and evoked activity of antidromically identified trigemino-thalamic tract (TGT) neurons was examined over the sleep-wake cycle in cats. There was no difference in the mean spike discharge rate of TGT neurons when quiet sleep (QS) and active sleep (AS) were compared with wakefulness (W). However, tooth pulp-evoked responses of TGT neurons were decreased during AS when compared to W. Conversely, the responses of TGT neurons to air puff activation of facial hair mechanoreceptors reciprocally increased during AS when compared to W. The present data demonstrate that ascending sensory information emanating from distinct orofacial areas is differentially modified during the behavioral state of AS. Specifically, the results obtained suggest that during AS, sensory information arising from hair mechanoreceptors is enhanced, whereas information arising from tooth pulp afferents is suppressed. These data may provide functional evidence for an AS-related gate control mechanism of sensory outflow to higher brain centers.

Functional classification of afferent phrenic nerve fibres and diaphragmatic receptors in cats.

1. Single afferent fibres with receptive fields in the diaphragm (272 units) dissected from the right phrenic nerve were classified according to the following properties: reaction to contraction of the diaphragm, resting activity, conduction velocity, location and properties of receptive fields, and reaction to injection of bradykinin and lactic acid into the internal thoracic artery. Nine additional fibres dissected from the phrenic nerve had receptive fields outside the diaphragm. The experiments were performed on chloralose-anaesthetized cats. 2. Ninety-six fibres (36%) had high resting activity when unloaded by contraction of the diaphragm, had low-threshold receptive fields in the muscle and were mostly group II and III fibres. They probably innervated muscle spindles. 3. Eighty-eight fibres (32%) were vigorously activated by contraction of the diaphragm. They had low-threshold receptive fields located in the musculotendinous border and central tendon. Their conduction velocity was in the range for group II and III fibres. We infer that they may innervate tendon organs. 4. Eighty-eight fibres (32%) were slightly affected or not affected by diaphragmatic contraction. They had low- and high-threshold receptive fields located mostly in the muscular part of the diaphragm, and negligible resting activity. Most of them were group III and IV afferent fibres and were activated when bradykinin and lactic acid were applied to their receptive fields. Possibly these low- and high-threshold receptors innervated diaphragmatic ergo- and nociceptors, respectively. 5. Sensory outflow from the diaphragm was found to be somatotopically organized, so that fibres with receptive fields in the sternocostal portion were predominantly located in the upper phrenic nerve root, and those with lumbar receptive fields were in the lower root. 6. It is concluded that the phrenic nerve contains fibres from several distinct classes of sensory receptors: muscle spindles, tendon organs, ergoceptors and nociceptors. The sensory diaphragmatic outflow to the spinal cord is somatotopically organized.

The active cochlea

Peter Dallas Auditory Physiology Laboratory (The Hugh Knowles Center) and Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208 The cochlea is a hydromechanical frequency analyzer located in the inner ear (Fig. 1 a). Its principal role is to perform a real- time spectral decomposition of the acoustic signal in producing a spatial frequency map. The frequency analysis may be un- derstood with the aid of Figure 1 b, which shows a straightened cochlea with a snapshot of its basilar membrane displaced in response to a single-frequency sound (pure tone). Upon delivery of an acoustic signal into the fluid-filled cochlea, the basilar membrane undergoes an oscillatory motion at the frequency of the sound, resulting in a wave traveling toward its distal end. The drawing shows an instantaneous view ofthis traveling wave. The wave is spatially confined along the length of the basilar membrane, and the location of its maximum amplitude is re- lated to the frequency of the sound. The higher the frequency, the more restricted the disturbance to the proximal end. The vibrating membrane supports the sense organ of hearing, the spiral organ of Corti (Fig. 2) which is deformed maximally in the region of the peak of the traveling wave. In this location, the sensory receptor cells of the organ of Corti receive maximal mechanical stimulation, transduce it into maximal electrical signals, and thus produce maximal afferent sensory outflow from the cochlea. Thus, mechanical frequency analysis is performed by matching particular frequencies with particular groups of auditory receptor cells and their nerve fibers. Understanding of frequency analysis in the inner ear pro- gressed through three main epochs. The first was dominated by Helmholtz’s suggestions that lightly damped, spatially ordered, mechanically resonant elements in the cochlea perform the spec- tral analysis (this period was reviewed by Wever, 1949). The second epoch, lasting from the late 1940s to the early 1970s was dominated by von Bekesy’s description of the traveling wave (von BtkCsy, 1960). We are now in the third epoch (an overview of its evolution is given in Dallos, 1988) during which a fundamentally different paradigm has emerged. According to this paradigm, von Btktsy’s traveling wave is boosted by a local electromechanical amplification process in which one of the mammalian ear’s sensory cell groups, outer hair cells, function as both sensors and mechanical feedback elements. The ideas that the operation of a sense organ is dependent upon local mechanical feedback modification by what resembles a sensory receptor cell, that such mechanical feedback may operate at audio frequencies utilizing some novel cellular motor, and that

Sympathetic and afferent somata projecting in hindlimb nerves and the anatomical organization of the lumbar sympathetic nervous system of the rat.

The anatomy of the sympathetic pathways from the spinal cord to the lumbar sympathetic trunk and the inferior mesenteric ganglion was studied systematically in the rat. Details of the arrangements of white and gray rami communicantes, sympathetic trunk ganglia, the intermesenteric nerve, and the lumbar splanchnic nerves are summarized. A modified nomenclature for the segmental ganglia of the paravertebral sympathetic chain is proposed. Cell bodies of sensory and sympathetic axons projecting to the skin and skeletal muscle of the rat hindlimb were labeled retrogradely with horseradish peroxidase (HRP) in order to study numbers, segmental distribution, and location of the somata of these neurons quantitatively. HRP was applied to the nerves supplying skeletal muscle (gastrocnemius-soleus, GS), hairy skin (sural, SU; saphenous, SA) and to a mixed nerve (tibial, TI). All sensory somata and 96.4% of the sympathetic cell bodies were located ipsilaterally. Sensory somata were commonly restricted to two adjacent dorsal root ganglia (usually L3-4 for SA; L4-5 for GS, TI; L5-6 for SU). Although the sympathetic somata were more widely distributed rostrocaudally (four to six segments), their maximum was always located one or two segments more cranially than the sensory outflow, i.e., corresponding to the rami communicantes grisei. From the data, it is estimated that 420 sympathetic and 530 afferent neurons project into GS, 590 and 3,610 into SU, 920 and 3,750 into SA, and 1,070 and 5,760 into TI. These absolute neuron numbers are compared with electron microscopic fiber counts from the literature.