Brett A. Graham

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.

Morphological, neurochemical and electrophysiological features of parvalbumin‐expressing cells: a likely source of axo‐axonic inputs in the mouse spinal dorsal horn

•  Perception of normal bodily sensations relies on the precise regulation of sensory information entering the dorsal horn of the spinal cord. •  Inhibitory, axoaxonic, synapses provide a mechanism for this regulation, but the source of these important inhibitory connections remains to be elucidated. •  This study shows that a subpopulation of spinal interneurons that expresses parvalbumin and have specific morphological, connectivity and functional characteristics are a likely source of the inhibitory inputs that selectivity regulate non‐noxious tactile input in the spinal cord. •  Our findings suggest that a loss of normal function in parvalbumin positive dorsal horn neurons may result in the development of tactile allodynia, where non‐painful stimuli gain the capacity to evoke the sensation of pain.

Distinct Physiological Mechanisms Underlie Altered Glycinergic Synaptic Transmission in the Murine Mutants spastic, spasmodic, and oscillator

Spastic (spa), spasmodic (spd), and oscillator (ot) mice have naturally occurring glycine receptor (GlyR) mutations, which manifest as motor deficits and an exaggerated “startle response.” Using whole-cell recording in hypoglossal motoneurons, we compared the physiological mechanisms by which each mutation alters GlyR function. Mean glycinergic miniature IPSC (mIPSC) amplitude and frequency were dramatically reduced (>50%) compared with controls for each mutant. mIPSC decay times were unchanged in spa/spa (4.5 ± 0.3 vs 4.7 ± 0.2 ms), reduced in spd/spd (2.7 ± 0.2 vs 4.7 ± 0.2 ms), and increased in ot/ot (12.3 ± 1.2 vs 4.8 ± 0.2 ms). Thus, in spastic, GlyRs are functionally normal but reduced in number, whereas in spasmodic, GlyR kinetics is faster. The oscillator mutation results in complete absence of α1-containing GlyRs; however, some non-α1-containing GlyRs persist at synapses. Fluctuation analysis of membrane current, induced by glycine application to outside-out patches, showed that mean single-channel conductance was increased in spa/spa (64.2 ± 4.9 vs 36.1 ± 1.4 pS), but unchanged in spd/spd (32.4 ± 2.1 vs 35.3 ± 2.1 pS). GlyR-mediated whole-cell currents in spa/spa exhibited increased picrotoxin sensitivity (27 vs 71% block for 100 μm), indicating α1 homomeric GlyR expression. The picrotoxin sensitivity of evoked glycinergic IPSCs and conductance of synaptic GlyRs, as determined by nonstationary variance analysis, were identical for spa/spa and controls. Together, these findings show the three mutations disrupt GlyR-mediated inhibition via different physiological mechanisms, and the spastic mutation results in “compensatory” α1 homomeric GlyRs at extrasynaptic loci.

Orexin antagonists for neuropsychiatric disease: progress and potential pitfalls

The tight regulation of sleep/wake states is critical for mental and physiological wellbeing. For example, dysregulation of sleep/wake systems predisposes individuals to metabolic disorders such as obesity and psychiatric problems, including depression. Contributing to this understanding, the last decade has seen significant advances in our appreciation of the complex interactions between brain systems that control the transition between sleep and wake states. Pivotal to our increased understanding of this pathway was the description of a group of neurons in the lateral hypothalamus (LH) that express the neuropeptides orexin A and B (hypocretin, Hcrt-1 and Hcrt-2). Orexin neurons were quickly placed at center stage with the demonstration that loss of normal orexin function is associated with the development of narcolepsy—a condition in which sufferers fail to maintain normal levels of daytime wakefulness. Since these initial seminal findings, much progress has been made in our understanding of the physiology and function of the orexin system. For example, the orexin system has been identified as a key modulator of autonomic and neuroendocrine function, arousal, reward and attention. Notably, studies in animals suggest that dysregulation of orexin function is associated with neuropsychiatric states such as addiction and mood disorders including depression and anxiety. This review discusses the progress associated with therapeutic attempts to restore orexin system function and treat neuropsychiatric conditions such as addiction, depression and anxiety. We also highlight potential pitfalls and challenges associated with targeting this system to treat these neuropsychiatric states.

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.

Cocaine potentiates excitatory drive in the perifornical/lateral hypothalamus

•  Drugs of addiction are well‐established in their capacity to alter brain reward pathways. •  The perifornical/lateral hypothalamus has previously been shown to be drug responsive, participate in relapse to drug taking, and project to key reward pathway structures. •  This study demonstrates that cocaine enhances excitatory drive to perifornical/lateral hypothalamic neurones, and these changes involve altered presynaptic function. Orexin‐positive neurones were among the populations that underwent these presynaptic changes. •  The results indicate that a greater understanding of the drug‐induced synaptic changes in perifornical/lateral hypothalamus may instruct future pharmacotherapies aimed at preventing drug relapse.

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.

Probing glycine receptor stoichiometry in superficial dorsal horn neurones using the spasmodic mouse

Non‐technical summary  Glycine receptors (GlyRs) play an important role in inhibiting neurone activity in the spinal cord. Until recently adult GlyRs were thought to comprise α1 and β subunits. A new form of the receptor containing α3 subunits has been discovered in the superficial dorsal horn (SDH), a region of the spinal cord important for pain. This raises questions about the precise subunit composition of GlyRs and glycinergic synapses in the SDH. We used the spasmodic mouse, where α1 subunit containing GlyRs have altered agonist sensitivity and electrophysiological properties, to ask how α1 and α3 subunits are assembled to form GlyRs on SDH neurones. We found most (∼75%) GlyRs and glycinergic synapses in the SDH contain α1 subunits and few are composed exclusively of α3 subunits. Therefore, future efforts to design pain drugs that target the α3 subunit must consider the potential interaction between α1 and α3 subunits in the GlyR.

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.