Properties of Deiters' neurons and inhibitory synaptic transmission in the mouse lateral vestibular nucleus.

Deiters' neurons, located exclusively in the lateral vestibular nucleus (LVN), are involved in vestibulospinal reflexes, innervate extensor motoneurons that drive antigravity muscles, and receive inhibitory inputs from the cerebellum. We investigated intrinsic membrane properties, short-term plasticity, and inhibitory synaptic inputs of mouse Deiters' and non-Deiters' neurons within the LVN. Deiters' neurons are distinguished from non-Deiters' neurons by their very low input resistance (105.8 vs. 521.8 MΩ, respectively), long axons that project as far as the ipsilateral lumbar spinal cord, and expression of the cytostructural protein nonphosphorylated neurofilament protein (NPNFP). Whole cell patch-clamp recordings in brain stem slices show that most Deiters' and non-Deiters' neurons were tonically active (>92%). Short-term plasticity was studied by examining discharge rate modulation following release from hyperpolarization [postinhibitory rebound firing (PRF)] and depolarization [firing rate adaptation (FRA)]. PRF and FRA gain were similar in Deiters' and non-Deiters' neurons (PRF 24.9 vs. 20.2 Hz and FRA gain 231.5 vs. 287.8 spikes/s/nA, respectively). Inhibitory synaptic input to both populations showed that GABAergic rather than glycinergic inhibition dominated. However, GABAA miniature inhibitory postsynaptic current (mIPSC) frequency was much higher in Deiters' neurons compared with non-Deiters' neurons (∼15.9 vs. 1.4 Hz, respectively). Our data suggest that Deiters' neurons can be reliably identified by their intrinsic membrane and synaptic properties. They are tonically active and glutamatergic, have low sensitivity or "gain," exhibit little adaptation, and receive strong GABAergic input. Deiters' neurons also have minimal short-term plasticity, and together these features suggest they are well suited to a role in encoding tonic signals for the vestibulospinal reflex.NEW & NOTEWORTHY Deiters' neurons within the lateral vestibular nucleus project the length of the spinal cord and activate antigravity extensor muscles. Deiters' neurons were characterized anatomically and physiologically in mice. Deiters' neurons are tonically active, have homogeneous intrinsic membrane properties, including low input resistance, and receive significant GABAAergic synaptic inputs. Deiters' neurons show little modulation in response to current injection. These features are consistent with Deiters' neurons responding to perturbations to maintain posture and balance.

A review of efferent cholinergic synaptic transmission in the vestibular periphery and its functional implications.

It has been over 60 years since peripheral efferent vestibular terminals were first identified in mammals, and yet the function of the efferent vestibular system remains obscure. One reason for the lack of progress may be due to our deficient understanding of the peripheral efferent synapse. Although vestibular efferent terminals were identified as cholinergic less than a decade after their anatomical characterization, the cellular mechanisms that underlie the properties of these synapses have had to be inferred. In this review we examine how recent mammalian studies have begun to reveal both nicotinic and muscarinic effects at these terminals and therefore provide a context for fast and slow responses observed in classic electrophysiological studies of the mammalian efferent vestibular system, nearly 40 years ago. Although incomplete, these new results together with those of recent behavioral studies are helping to unravel the mysterious and perplexing action of the efferent vestibular system. Armed with this information, we may finally appreciate the behavioral framework in which the efferent vestibular system operates.

Intrinsic excitability differs between murine hypoglossal and spinal motoneurons.

Motoneurons differ in the behaviors they control and their vulnerability to disease and aging. For example, brain stem motoneurons such as hypoglossal motoneurons (HMs) are involved in licking, suckling, swallowing, respiration, and vocalization. In contrast, spinal motoneurons (SMs) innervating the limbs are involved in postural and locomotor tasks requiring higher loads and lower movement velocities. Surprisingly, the properties of these two motoneuron pools have not been directly compared, even though studies on HMs predominate in the literature compared with SMs, especially for adult animals. Here we used whole cell patch-clamp recording to compare the electrophysiological properties of HMs and SMs in age-matched neonatal mice (P7-P10). Passive membrane properties were remarkably similar in HMs and SMs, and afterhyperpolarization properties did not differ markedly between the two populations. HMs had narrower action potentials (APs) and a faster upstroke on their APs compared with SMs. Furthermore, HMs discharged APs at higher frequencies in response to both step and ramp current injection than SMs. Therefore, while HMs and SMs have similar passive properties, they differ in their response to similar levels of depolarizing current. This suggests that each population possesses differing suites of ion channels that allow them to discharge at rates matched to the different mechanical properties of the muscle fibers that drive their distinct motor functions.

In vivo characterization of colorectal and cutaneous inputs to lumbosacral dorsal horn neurons in the mouse spinal cord.

Chronic abdominal pain is a common symptom of inflammatory bowel disease and often persists in the absence of gut inflammation. Although the mechanisms responsible for ongoing pain are unknown, clinical and preclinical evidence suggests lumbosacral spinal cord dorsal horn neurons contribute to these symptoms. At present, we know little about the intrinsic and synaptic properties of this population of neurons in either normal or inflammed conditions. Therefore, we developed an in vivo preparation to make patch-clamp recordings from superficial dorsal horn (SDH) neurons receiving colonic inputs in naïve male mice. Recordings were made in the lumbosacral spinal cord (L6-S1) under isoflurane anesthesia. Noxious colorectal distension (CRD) was used to determine whether SDH neurons received inputs from mechanical stimulation/distension of the colon. Responses to hind paw/tail cutaneous stimulation and intrinsic and synaptic properties were also assessed, as well as action potential discharge properties. Approximately 11% of lumbosacral SDH neurons in the cohort of neurons sampled responded to CRD and a majority of these responses were subthreshold. Most CRD-responsive neurons (80%) also responded to cutaneous stimuli, compared with <50% of CRD-non-responsive neurons. Furthermore, CRD-responsive neurons had more hyperpolarized resting membrane potentials, larger rheobase currents, and reduced levels of excitatory drive, compared to CRD-non-responsive neurons. Our results demonstrate that CRD-responsive neurons can be distinguished from CRD-non-responsive neurons by several differences in their membrane properties and excitatory synaptic inputs. We also demonstrate that SDH neurons with colonic inputs show predominately subthreshold responses to CRD and exhibit a high degree of viscerosomatic convergence.

Electrical maturation of spinal neurons in the human fetus: comparison of ventral and dorsal horn.

The spinal cord is critical for modifying and relaying sensory information to, and motor commands from, higher centers in the central nervous system to initiate and maintain contextually relevant locomotor responses. Our understanding of how spinal sensorimotor circuits are established during in utero development is based largely on studies in rodents. In contrast, there is little functional data on the development of sensory and motor systems in humans. Here, we use patch-clamp electrophysiology to examine the development of neuronal excitability in human fetal spinal cords (10-18 wk gestation; WG). Transverse spinal cord slices (300 µm thick) were prepared, and recordings were made, from visualized neurons in either the ventral (VH) or dorsal horn (DH) at 32°C. Action potentials (APs) could be elicited in VH neurons throughout the period examined, but only after 16 WG in DH neurons. At this age, VH neurons discharged multiple APs, whereas most DH neurons discharged single APs. In addition, at 16-18 WG, VH neurons also displayed larger AP and after-hyperpolarization amplitudes than DH neurons. Between 10 and 18 WG, the intrinsic properties of VH neurons changed markedly, with input resistance decreasing and AP and after-hyperpolarization amplitudes increasing. These findings are consistent with the hypothesis that VH motor circuitry matures more rapidly than the DH circuits that are involved in processing tactile and nociceptive information.

Intrinsic and synaptic homeostatic plasticity in motoneurons from mice with glycine receptor mutations.

Inhibitory synaptic inputs to hypoglossal motoneurons (HMs) are important for modulating excitability in brainstem circuits. Here we ask whether reduced inhibition, as occurs in three murine mutants with distinct naturally occurring mutations in the glycine receptor (GlyR), leads to intrinsic and/or synaptic homeostatic plasticity. Whole cell recordings were obtained from HMs in transverse brainstem slices from wild-type (wt), spasmodic (spd), spastic (spa), and oscillator (ot) mice (C57Bl/6, approximately postnatal day 21). Passive and action potential (AP) properties in spd and ot HMs were similar to wt. In contrast, spa HMs had lower input resistances, more depolarized resting membrane potentials, higher rheobase currents, smaller AP amplitudes, and slower afterhyperpolarization current decay times. The excitability of HMs, assessed by "gain" in injected current/firing-frequency plots, was similar in all strains whereas the incidence of rebound spiking was increased in spd. The difference between recruitment and derecruitment current (i.e., ΔI) for AP discharge during ramp current injection was more negative in spa and ot. GABAA miniature inhibitory postsynaptic current (mIPSC) amplitude was increased in spa and ot but not spd, suggesting diminished glycinergic drive leads to compensatory adjustments in the other major fast inhibitory synaptic transmitter system in these mutants. Overall, our data suggest long-term reduction in glycinergic drive to HMs results in changes in intrinsic and synaptic properties that are consistent with homeostatic plasticity in spa and ot but not in spd. We propose such plasticity is an attempt to stabilize HM output, which succeeds in spa but fails in ot.

Are all spinal segments equal: intrinsic membrane properties of superficial dorsal horn neurons in the developing and mature mouse spinal cord.

Neurons in the superficial dorsal horn (SDH; laminae I-II) of the spinal cord process nociceptive information from skin, muscle, joints and viscera. Most of what we know about the intrinsic properties of SDH neurons comes from studies in lumbar segments of the cord even though clinical evidence suggests nociceptive signals from viscera and head and neck tissues are processed differently. This 'lumbar-centric' view of spinal pain processing mechanisms also applies to developing SDH neurons. Here we ask whether the intrinsic membrane properties of SDH neurons differ across spinal cord segments in both the developing and mature spinal cord. Whole cell recordings were made from SDH neurons in slices of upper cervical (C2-4), thoracic (T8-10) and lumbar (L3-5) segments in neonatal (P0-5) and adult (P24-45) mice. Neuronal input resistance (R(IN)), resting membrane potential, AP amplitude, half-width and AHP amplitude were similar across spinal cord regions in both neonates and adults (∼100 neurons for each region and age). In contrast, these intrinsic membrane properties differed dramatically between neonates and adults. Five types of AP discharge were observed during depolarizing current injection. In neonates, single spiking dominated (∼40%) and the proportions of each discharge category did not differ across spinal regions. In adults, initial bursting dominated in each spinal region, but was significantly more prevalent in rostral segments (49% of neurons in C2-4 vs. 29% in L3-5). During development the dominant AP discharge pattern changed from single spiking to initial bursting. The rapid A-type potassium current (I(Ar)) dominated in neonates and adults, but its prevalence decreased (∼80% vs. ∼50% of neurons) in all regions during development. I(Ar) steady state inactivation and activation also changed in upper cervical and lumbar regions during development. Together, our data show the intrinsic properties of SDH neurons are generally conserved in the three spinal cord regions examined in both neonate and adult mice. We propose the conserved intrinsic membrane properties of SDH neurons along the length of the spinal cord cannot explain the marked differences in pain experienced in the limbs, viscera, and head and neck.

Attenuated glycine receptor function reduces excitability of mouse medial vestibular nucleus neurons.

Spontaneous activity in medial vestibular nucleus (MVN) neurons is modulated by synaptic inputs. These inputs are crucial for maintaining gaze and posture and contribute to vestibular compensation after lesions of peripheral vestibular organs. We investigated how chronically attenuated glycinergic input affects excitability of MVN neurons. To this end we used three mouse strains (spastic, spasmodic, and oscillator), with well-characterized naturally occurring mutations in the inhibitory glycine receptor (GlyR). First, using whole-cell patch-clamp recordings, we demonstrated that the amplitude of the response to rapidly applied glycine was dramatically reduced by 25 to 90% in MVN neurons from mutant mice. We next determined how reduced GlyR function affected MVN neuron output. Neurons were classified using two schemas: (1) the shape of their action potential afterhyperpolarization (AHP); and (2) responses to hyperpolarizing current injection. In the first schema, neurons were classified as types A, B and C. The prevalence of type C neurons in the mutant strains was significantly increased. In the second schema, the proportion of neurons lacking post inhibitory rebound firing (PRF-deficient) was increased. In both schemas an increase in AHP amplitude was a common feature of the augmented neuron group (type C, PRF-deficient) in the mutant strains. We suggest increased AHP amplitude reduces overall excitability in the MVN and thus maintains network function in an environment of reduced glycinergic input.

Pacemaker currents in mouse locus coeruleus neurons.

We have characterized the currents that flow during the interspike interval in mouse locus coeruleus (LC) neurons, by application of depolarizing ramps and pulses, and compared our results with information available for rats. A tetrodotoxin (TTX)-sensitive current was the only inward conductance active during the interspike interval; no TTX-insensitive Na(+) or oscillatory currents were detected. Ca(2+)-free and Ba(2+)-containing solutions failed to demonstrate a Ca(2+) current during the interspike interval, although a Ca(2+) current was activated at membrane potentials positive to -40 mV. A high- tetraethylammonium chloride (TEA) (15 mM) sensitive current accounted for almost all the K(+) conductance during the interspike interval. Ca(2+)-activated K(+), inward rectifier and low-TEA (10 muM) sensitive currents were not detected within the interspike interval. Comparison of these findings to those reported for neonatal rat LC neurons indicates that the pacemaker currents are similar, but not identical, in the two species with mice lacking a persistent Ca(2+) current during the interspike interval. The net pacemaking current determined by differentiating the interspike interval from averaged action potential recordings closely matched the net ramp-induced currents obtained either under voltage clamp or after reconstructing this current from pharmacologically isolated currents. In summary, our results suggest the interspike interval pacemaker mechanism in mouse LC neurons involves a combination of a TTX-sensitive Na(+) current and a high TEA-sensitive K(+) current. In contrast with rats, a persistent Ca(2+) current is not involved.

Evidence for a critical period in the development of excitability and potassium currents in mouse lumbar superficial dorsal horn neurons.

The output of superficial dorsal horn (SDH; laminae I-II) neurons is critical for processing nociceptive, thermal, and tactile information. Like other neurons, the combined effects of synaptic inputs and intrinsic membrane properties determine their output. It is well established that peripheral synaptic inputs to SDH neurons undergo extensive reorganization during pre- and postnatal development. It is unclear, however, how membrane properties or the subthreshold whole cell currents that shape SDH neuron output change during this period. Here we assess the intrinsic membrane properties and whole cell currents in mouse SDH neurons during late embryonic and early postnatal development (E15-P25). Transverse slices were prepared from lumbar spinal cord and whole cell recordings were obtained at 32 degrees C. During this developmental period resting membrane potential (RMP) became more hyperpolarized (by approximately 10 mV, E15-E17 vs. P21-P25) and input resistance decreased (1,074 +/- 78 vs. 420 +/- 27 MOmega). In addition, action potential (AP) amplitude and AP afterhyperpolarization increased, whereas AP half-width decreased. Before and after birth (E15-P10), AP discharge evoked by intracellular current injection was limited to a single AP at depolarization onset in many neurons (>41%). In older animals (P11-P25) this changed, with AP discharge consisting of brief bursts at current onset ( approximately 46% of neurons). Investigation of major subthreshold whole cell currents showed the rapid A-type potassium current (I(Ar)) dominated at all ages examined (90% of neurons at E15-E17, decreasing to >50% after P10). I(Ar) expression levels, based on peak current amplitude, increased during development. Steady-state inactivation and activation for I(Ar) were slightly less potent in E15-E17 versus P21-P25 neurons at potentials near RMP (-55 mV). Together, our data indicate that intrinsic properties and I(Ar) expression change dramatically in SDH neurons during development, with the greatest alterations occurring on either side of a critical period, P6-P10.

Recording temperature affects the excitability of mouse superficial dorsal horn neurons, in vitro.

Superficial dorsal horn (SDH) neurons in laminae I-II of the spinal cord play an important role in processing noxious stimuli. These neurons represent a heterogeneous population and are divided into various categories according to their action potential (AP) discharge during depolarizing current injection. We recently developed an in vivo mouse preparation to examine functional aspects of nociceptive processing and AP discharge in SDH neurons and to extend investigation of pain mechanisms to the genetic level of analysis. Not surprisingly, some in vivo data obtained at body temperature (37 degrees C) differed from those generated at room temperature (22 degrees C) in spinal cord slices. In the current study we examine how temperature influences SDH neuron properties by making recordings at 22 and 32 degrees C in transverse spinal cord slices prepared from L3-L5 segments of adult mice (C57Bl/6). Patch-clamp recordings (KCH(3)SO(4) internal) were made from visualized SDH neurons. At elevated temperature all SDH neurons had reduced input resistance and smaller, briefer APs. Resting membrane potential and AP afterhyperpolarization amplitude were temperature sensitive only in subsets of the SDH population. Notably, elevated temperature increased the prevalence of neurons that did not discharge APs during current injection. These reluctant firing neurons expressed a rapid A-type potassium current, which is enhanced at higher temperatures and thus restrains AP discharge. When compared with previously published whole cell recordings obtained in vivo (37 degrees C) our results suggest that, on balance, in vitro data collected at elevated temperature more closely resemble data collected under in vivo conditions.

Altered potassium channel function in the superficial dorsal horn of the spastic mouse.

The spastic mouse has a naturally occurring glycine receptor (GlyR) mutation that disrupts synaptic input in both motor and sensory pathways. Here we use the spastic mouse to examine how this altered inhibitory drive affects neuronal intrinsic membrane properties and signal processing in the superficial dorsal horn (SDH), where GlyRs contribute to pain processing mechanisms. We first used in vitro patch clamp recording in spinal cord slices (L3-L5 segments) to examine intrinsic membrane properties of SDH neurones in spastic and age-matched wildtype controls ( approximately P23). Apart from a modest reduction ( approximately 3 mV) in resting membrane potential (RMP), neurones in spastic mice have membrane and action potential (AP) properties identical to wildtype controls. There was, however, a substantial reorganization of AP discharge properties in neurones from spastic mice, with a significant increase (14%) in the proportion of delayed firing neurones. This was accompanied by a change in the voltage sensitivity of rapid A-currents, a possible mechanism for increased delayed firing. To assess the functional consequences of these changes, we made in vivo patch-clamp recordings from SDH neurones in urethane anaesthetized (2.2 g kg(-1), i.p.) spastic and wildtype mice ( approximately P37), and examined responses to innocuous and noxious mechanical stimulation of the hindpaw. Overall, responses recorded in wildtype and spastic mice were similar; however, in spastic mice a small population of spontaneously active neurones ( approximately 10%) exhibited elevated spontaneous discharge frequency and post-pinch discharge rates. Together, these results are consistent with the altered intrinsic membrane properties of SDH neurones observed in vitro having functional consequences for pain processing mechanisms in the spastic mouse in vivo. We propose that alterations in potassium channel function in the spastic mouse compensate, in part, for reduced glycinergic inhibition and thus maintain normal signal processing in the SDH.

Moving from an averaged to specific view of spinal cord pain processing circuits.

Neurons in the superficial dorsal horn (SDH) of the spinal cord play a critical role in processing potentially painful or noxious signals from skin, muscle, and viscera. Many acute pain therapies are based on the notion that altering the excitability of SDH neurons can block or gate these signals and reduce pain. This same notion also underlies treatments for certain chronic pain states. Basic scientists are now beginning to identify a number of potential molecular targets for spinal cord-based pain therapies with a focus on ion channels and receptors that can alter neuronal excitability. The current challenge in pain research is to identify which are the most promising targets and how their manipulation alters pain processing. In this review, we propose that our understanding of spinal pain processing mechanisms and translation of these discoveries into pain therapies could be improved by 1) better appreciating and understanding neuronal heterogeneity in the SDH; 2) establishing connectivity patterns among SDH neuron types; and 3) testing and extending findings made in vitro to intact (in vivo) animal models. As this information becomes available, it will be possible to determine the precise distribution of potential therapeutic targets on various SDH neuron types within specific circuits known to be functionally important in spinal pain processing.

Pinch-current injection defines two discharge profiles in mouse superficial dorsal horn neurones, in vitro.

Neurones in the superficial dorsal horn (SDH) are a major target for nociceptive afferents and play an important role in pain processing. One approach to understanding the role of SDH neurones has been to study their action potential (AP) discharge in spinal cord slices during injection of depolarizing step-currents. Four or five neurone subpopulations are typically identified based on AP discharge, with various roles proposed for each in pain processing. During noxious peripheral stimulation in vivo, however, SDH neurones are activated via synaptic inputs. This produces a conductance change with different somato-dendritic distributions and temporal characteristics to that provided by a somatic step-current injection. Here we introduce an alternative approach to studying SDH neurone discharge under in vitro conditions. We recorded voltage-clamp responses in SDH neurones, in vivo, during noxious mechanical stimulation of the hindpaw (1 s pinch, approximately 100 g mm(-2)). From these recordings a representative 'pinch-current' was selected and subsequently injected into SDH neurones in spinal cord slices (recording temperature 32 degrees C). Pinch-current-evoked discharge was compared to that evoked by rectangular step-current injections. Pinch- and step-current-evoked AP discharge frequency was highly correlated (r2 = 0.61). This was also true for rheobase current comparisons (r2 = 0.61). Conversely, latency to discharge and discharge duration were not correlated when step- and pinch-current responses were compared. When neurones were grouped according to step-current-evoked discharge, five distinct patterns were apparent (tonic firing, initial bursting, delayed firing, single spiking, and reluctant firing). In contrast, pinch-current responses separated into two clear patterns of activity (robust and resistant firing). During pinch-current injection, tonic-firing and initial-bursting neurones exhibited robust AP discharge with similar characteristics. In contrast, single-spiking and reluctant-firing neurones were resistant to AP discharge. Delayed-firing neurones exhibited pinch-current responses that were transitional between those of tonic-firing/initial-bursting and single-spiking/reluctant-firing neurones. Injection of digitally filtered pinch-currents indicated that transient current fluctuations are necessary for robust repetitive discharge in initial-bursting neurones. These data suggest the functional significance of the diverse step-current-evoked firing patterns, previously reported in SDH neurones remains to be fully understood. When a 'facsimile' current profile or pinch-current is used in place of step-currents, AP discharge diversity is much reduced.

In vivo responses of mouse superficial dorsal horn neurones to both current injection and peripheral cutaneous stimulation.

In the superficial dorsal horn (SDH) processing of noxious and innocuous stimuli is critically dependent on the input-output relationship of its component neurones. Such relationships are routinely examined by assessing neuronal responses to somatic current injection or activation of synaptic inputs. A more complete understanding of input-output relationships would be achieved by comparing, in the same neurone, how the two forms of activation contribute to neuronal output. Therefore, we examined how SDH neurones transform depolarizing current injections and synaptic excitation via peripheral cutaneous stimuli (brush and pinch of the hindpaw) into trains of action potentials, in an in vivo preparation of the adult mouse spinal cord. Under whole-cell current clamp recording conditions four action potential discharge patterns were observed during depolarizing current injection: tonic firing neurones (21/93) discharged spikes throughout the step; initial bursting neurones (35/93) discharged several spikes at step onset; single spiking neurones (16/93) discharged one or two spikes at step onset; and delayed firing neurones (21/93) discharged spikes delayed from the step onset. Four characteristic profiles were observed in response to application of noxious (pinch) and innocuous (brush) cutaneous stimuli: nociceptive neurones (20/37) responded maximally to pinch stimulation; light touch neurones (9/37) responded maximally to brush stimulation; subthreshold neurones (4/37) exhibited depolarizing responses without firing action potentials; and hyperpolarizing neurones (4/37) exhibited a sustained pinch-induced hyperpolarization. Comparisons of current-evoked discharge patterns with peripherally evoked responses indicate SDH neurones expressing each of the four discharge patterns could receive, and therefore participate in the processing of information concerning, either noxious or innocuous stimuli. These data suggest that a neurone's response to current injection does not necessarily help identify or predict how the same neurone will respond to physiologically or functionally relevant stimuli.

An in vivo mouse spinal cord preparation for patch-clamp analysis of nociceptive processing.

The laboratory mouse is now considered the preferred mammalian species for molecular and genetic analysis in neurobiology. In part, this is due to the existence, in the mouse, of several well characterised naturally occurring mutations in ligand gated ion channels and recent knockout, knockin, and transgenic techniques, which facilitate the manipulation of key molecules. These techniques have recently been applied to pain research with in vitro electrophysiological and behavioural techniques traditionally developed for the rat, now being adapted for the mouse particularly at the level of the spinal cord. Here, we describe an in vivo preparation of the mouse spinal cord for patch-clamp recording of nociceptive processing in the superficial dorsal horn (SDH) that permits analysis in the intact nervous system. We have recorded from SDH neurons and characterised their background synaptic activity, discharge properties, and evoked synaptic responses following controlled application of innocuous and noxious stimuli to the hind paw. Application of these techniques along with genetic, biomolecular, in vitro and behavioural approaches will allow future studies to comprehensively analyse the contributions of specific molecules involved in nociceptive processing in the spinal cord of a single species.

Morphological identification of physiologically characterized afferents innervating the turtle posterior crista.

The turtle posterior crista consists of two hemicristae. Each hemicrista extends from the planum semilunatum to the nonsensory torus and includes a central zone (CZ) surrounded by a peripheral zone (PZ). Type I and type II hair cells are found in the CZ and are innervated by calyx, dimorphic and bouton afferents. Only type II hair cells and bouton fibers are found in the PZ. Units were intraaxonally labeled in a half-head preparation. Bouton (B) units could be near the planum (BP), near the torus (BT), or in midportions of a hemicrista, including the PZ and CZ. Discharge properties of B units vary with longitudinal position in a hemicrista but not with morphological features of their peripheral terminations. BP units are regularly discharging and have small gains and small phase leads re angular head velocity. BT units are irregular and have large gains and large phase leads. BM units have intermediate properties. Calyx (C) and dimorphic (D) units have similar discharge properties and were placed into a single calyx-bearing (CD) category. While having an irregular discharge resembling BT units, CD units have gains and phases similar to those of BM units. Rather than any single discharge property, it is the relation between discharge regularity and either gain or phase that makes CD units distinctive. Multivariate statistical formulas were developed to infer a unit's morphological class (B or CD) and longitudinal position solely from its discharge properties. To verify the use of the formulas, discharge properties were compared for units recorded intraaxonally or extracellularly in the half-head or extracellularly in intact animals. Most B units have background rates of 10-30 spikes/s. The CD category was separated into CD-high and CD-low units with background rates above or below 5 spikes/s, respectively. CD-low units have lower gains and phases and are located nearer the planum than CD-high units. In their response dynamics over a frequency range from 0.01-3 Hz, BP units conform to an overdamped torsion-pendulum model. Other units show departures from the model, including high-frequency gain increases and phase leads. The longitudinal gradient in the physiology of turtle B units resembles a similar gradient in the anamniote crista. In many respects, turtle CD units have discharge properties resembling those of calyx-bearing units in the mammalian central zone.

Responses to efferent activation and excitatory response-intensity relations of turtle posterior-crista afferents.

Multivariate statistical formulas were used to infer the morphological type and longitudinal position of extracellularly recorded afferents. Efferent fibers were stimulated electrically in the nerve branch interconnecting the anterior and posterior VIIIth nerves. Responses of bouton (B) units depended on their inferred position: BP units (near the planum semilunatum) showed small excitatory responses; BT units (near the torus) were inhibited; BM units (in an intermediate position) had a mixed response, including an initial inhibition and a delayed excitation. Calyx-bearing (CD-high) units with an appreciable background discharge showed large per-train excitatory responses followed by smaller post-train responses that could outlast the shock train by 100 s. Excitatory responses were smaller in calyx-bearing (CD-low) units having little or no background activity than in CD-high units. Excitatory response-intensity functions, derived from the discharge during 2-s angular-velocity ramps varying in intensity, were fit by empirical functions that gave estimates of the maximal response (r(MAX)), a threshold velocity (v(T)), and the velocity producing a half-maximal response (v(1/2)). Linear gain is equal to r(MAX)/v(S), v(S) = v(1/2) - v(T). v(S) provides a measure of the velocity range over which the response is nearly linear. For B units, r(MAX) declines by as much as twofold over the 2-s ramp, whereas for CD units, r(MAX) increases by 15% during the same time period. At the end of the ramp, r(MAX) is on average twice as high in CD as in B units. Thresholds are negligible in most spontaneously active units, including almost all B and CD-high units. Silent CD-low units typically have thresholds of 10-100 deg/s. BT units have very high linear gains and v(S) < 10 deg/s. Linear gains are considerably lower in BP units and v(S) > 150 deg/s. CD-high units have intermediate gains and near 100 deg/s v(S) values. CD-low units have low gains and v(S) values ranging from 150 to more than 300 deg/s. The results suggest that BT units are designed to measure the small head movements involved in postural control, whereas BP and CD units are more appropriate for monitoring large volitional head movements. The former units are silenced by efferent activation, whereas the latter units are excited. This suggests that the efferent system switches the turtle posterior crista from a "postural" to a "volitional" mode.

Topographic distribution of nicotinic acetylcholine receptors in the cristae of a turtle.

The neurochemical basis of cholinergic efferent modulation of afferent function in the vestibular periphery remains incompletely understood; however, there is cellular, biochemical and molecular biological evidence for both muscarinic and nicotinic acetylcholine (ACh) receptors (nAChRs) in this system. This study examined the topographic distribution of alpha-bungarotoxin (alpha-BTX) nAChRs in the cristae of a turtle species. Cristae were perfusion-fixed, cut at 20 micrometer on a cryostat and incubated with alpha-BTX or polyclonal antibodies raised against Torpedo nAChR. Light microscopy showed abundant specific labeling of nAChR in the central zone of each hemicrista on the calyx-bearing afferents surrounding type I hair cells and on the base of the type II hair cells. Within the peripheral zone, dense labeling of type II hair cells near the torus and sparse or no label was observed on type II hair cells near the planum. The alpha-BTX binding showed a similar pattern within the cristae. The similarity between the topographic distribution of alpha-BTX binding nAChR and of efferent inhibition of afferents supports the notion that the inhibitory effect of afferents is mediated by nAChR.

Dizocilpine attenuates streptomycin-induced vestibulotoxicity in rats.

NMDA receptor mediated excitotoxicity contributes substantially to aminoglycoside antibiotic-induced cochlear damage. Since vestibular as well as cochlear hair cells have glutamatergic synapses, aminoglycoside-induced vestibulotoxicity may also have an excitotoxic component. This hypothesis was tested by examining the effects of the uncompetitive NMDA receptor antagonist dizocilpine on streptomycin-induced vestibulotoxicity. Streptomycin-treated rats exhibited almost complete destruction of sensory hair cells in the crista ampullaris, vestibular impairment in the drop test, and hyperkinesia. Concurrent treatment with dizocilpine not only rescued a substantial population of sensory hair cells in the cristae, but prevented the attendant hyperkinesis and vestibular impairments. These results indicate that excitotoxic mechanisms contribute to aminoglycoside-induced vestibulotoxicity and that NMDA antagonists may be useful in attenuating aminoglycoside ototoxicity.