Alan M. Brichta

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 neurones response to current injection does not necessarily help identify or predict how the same neurone will respond to physiologically or functionally relevant stimuli.

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.

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.

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.

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, ∼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°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.

Planar Relations of Semicircular Canals in Awake, Resting Turtles, Pseudemys scripta

As part of a project aimed at elucidating mechanisms of vestibulocollic control in the red-eared turtle Pseudemys scripta, we have calculated the planar relations of its semicircular canals using principal-components analysis. This information is prerequisite to understanding the pattern of canal activation that is set up by head movement of any spatial form. In addition, we have developed a method for monitoring canal orientation in an awake, behaving animal, and we have used this technique to assess canal position in resting turtles. Our results indicate that ipsilateral canals in Pseudemys are not mutually orthogonal, nor are complementary canals precisely coplanar, although they approach this idealized condition more closely than do the canals of several other vertebrates for which quantitative data exist. One significant departure from the perfectly orthogonal configuration is that both verical canals are rotated slightly toward the frontal plane; thus, Pseudemys should be somewhat more sensitive to head roll than to head rotation in other planes. Radiographic analyses of awake, resting turtles indicate that the anterior interparietal suture is held aligned with the earth horizontal and midsagittal plane. The horizontal canal is pitched up (open anterior) 3-4 degrees relative to the earth horizontal.

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 (∼P23). Apart from a modest reduction (∼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 (∼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 (∼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.

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.

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.

Widespread Vestibular Activation of the Rodent Cortex

Much of our understanding of the neuronal mechanisms of spatial navigation is derived from chronic recordings in rodents in which head-direction, place, and grid cells have all been described. However, despite the proposed importance of self-reference information to these internal representations of space, their congruence with vestibular signaling remains unclear. Here we have undertaken brain-wide functional mapping using both fMRI and electrophysiological methods to directly determine the spatial extent, strength, and time course of vestibular signaling across the rat forebrain. We find distributed activity throughout thalamic, limbic, and particularly primary sensory cortical areas in addition to known head-direction pathways. We also observe activation of frontal regions, including infralimbic and cingulate cortices, indicating integration of vestibular information throughout functionally diverse cortical regions. These whole-brain activity maps therefore suggest a widespread contribution of vestibular signaling to a self-centered framework for multimodal sensorimotor integration in support of movement planning, execution, spatial navigation, and autonomic responses to gravito-inertial changes.