Heteromeric α/ß glycine receptors regulate excitability in parvalbumin-expressing dorsal horn neurons through phasic and tonic glycinergic inhibition.

KEY POINTS: Spinal parvalbumin-expressing interneurons have been identified as a critical source of inhibition to regulate sensory thresholds by gating mechanical inputs in the dorsal horn. This study assessed the inhibitory regulation of the parvalbumin-expressing interneurons, showing that synaptic and tonic glycinergic currents dominate, blocking neuronal or glial glycine transporters enhances tonic glycinergic currents, and these manipulations reduce excitability. Synaptically released glycine also enhanced tonic glycinergic currents and resulted in decreased parvalbumin-expressing interneuron excitability. Analysis of the glycine receptor properties mediating inhibition of parvalbumin neurons, as well as single channel recordings, indicates that heteromeric α/ß subunit-containing receptors underlie both synaptic and tonic glycinergic currents. Our findings indicate that glycinergic inhibition provides critical control of excitability in parvalbumin-expressing interneurons in the dorsal horn and represents a pharmacological target to manipulate spinal sensory processing. ABSTRACT: The dorsal horn (DH) of the spinal cord is an important site for modality-specific processing of sensory information and is essential for contextually relevant sensory experience. Parvalbumin-expressing inhibitory interneurons (PV+ INs) have functional properties and connectivity that enables them to segregate tactile and nociceptive information. Here we examine inhibitory drive to PV+ INs using targeted patch-clamp recording in spinal cord slices from adult transgenic mice that express enhanced green fluorescent protein in PV+ INs. Analysis of inhibitory synaptic currents showed glycinergic transmission is the dominant form of phasic inhibition to PV+ INs. In addition, PV+ INs expressed robust glycine-mediated tonic currents; however, we found no evidence for tonic GABAergic currents. Manipulation of extracellular glycine by blocking either, or both, the glial and neuronal glycine transporters markedly decreased PV+ IN excitability, as assessed by action potential discharge. This decreased excitability was replicated when tonic glycinergic currents were increased by electrically activating glycinergic synapses. Finally, we show that both phasic and tonic forms of glycinergic inhibition are mediated by heteromeric α/ß glycine receptors. This differs from GABAA receptors in the dorsal horn, where different receptor stoichiometries underlie phasic and tonic inhibition. Together these data suggest both phasic and tonic glycinergic inhibition regulate the output of PV+ INs and contribute to the processing and segregation of tactile and nociceptive information. The shared stoichiometry for phasic and tonic glycine receptors suggests pharmacology is unlikely to be able to selectively target each form of inhibition in PV+ INs.

Distinct forms of synaptic inhibition and neuromodulation regulate calretinin-positive neuron excitability in the spinal cord dorsal horn.

The dorsal horn (DH) of the spinal cord contains a heterogenous population of neurons that process incoming sensory signals before information ascends to the brain. We have recently characterized calretinin-expressing (CR+) neurons in the DH and shown that they can be divided into excitatory and inhibitory subpopulations. The excitatory population receives high-frequency excitatory synaptic input and expresses delayed firing action potential discharge, whereas the inhibitory population receives weak excitatory drive and exhibits tonic or initial bursting discharge. Here, we characterize inhibitory synaptic input and neuromodulation in the two CR+ populations, in order to determine how each is regulated. We show that excitatory CR+ neurons receive mixed inhibition from GABAergic and glycinergic sources, whereas inhibitory CR+ neurons receive inhibition, which is dominated by glycine. Noradrenaline and serotonin produced robust outward currents in excitatory CR+ neurons, predicting an inhibitory action on these neurons, but neither neuromodulator produced a response in CR+ inhibitory neurons. In contrast, enkephalin (along with selective mu and delta opioid receptor agonists) produced outward currents in inhibitory CR+ neurons, consistent with an inhibitory action but did not affect the excitatory CR+ population. Our findings show that the pharmacology of inhibitory inputs and neuromodulator actions on CR+ cells, along with their excitatory inputs can define these two subpopulations further, and this could be exploited to modulate discrete aspects of sensory processing selectively in the DH.

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.

Functional heterogeneity of calretinin-expressing neurons in the mouse superficial dorsal horn: implications for spinal pain processing.

KEY POINTS: The superficial spinal dorsal horn contains a heterogeneous population of neurons that process sensory inputs. Information on the properties of excitatory interneurons in this region is limited. As calretinin is a protein thought to be restricted to an excitatory population in this region, the aim of this study was to characterize calretinin-expressing neurons. Most calretinin cells (85%) exhibited large A-type potassium currents and delayed firing action potential discharge, and received strong excitatory synaptic input, whereas the remainder exhibited hyperpolarization-activated cation currents and low threshold T-type calcium currents, and tonic- or initial bursting firing patterns, and received weak excitatory synaptic input. These respective features are consistent with properties of excitatory and inhibitory interneuron populations in this region of the spinal cord. Our findings have resolved a previously unidentified population of inhibitory interneurons. Furthermore, the contrasting excitability patterns of excitatory and inhibitory calretinin-expressing neurons suggest that they play distinct roles in spinal sensory processing circuits. ABSTRACT: Neurons in the superficial dorsal horn (SDH) of the spinal cord play an important role in nociceptive, thermal, itch and light touch sensations. Excitatory interneurons comprise ∼65% of all SDH neurons but surprisingly few studies have investigated their role in spinal sensory processing. Here we use a transgenic mouse to study putative excitatory SDH neurons that express the calcium binding protein calretinin (CR). Our immunocytochemical, morphological and electrophysiological analysis identified two distinct populations of CR-expressing neurons, which we termed 'Typical' and 'Atypical'. Typical CR-expressing neurons comprised ∼85% of the population and exhibited characteristic excitatory interneuron properties including delayed firing discharge, large rapid A-type potassium currents, and central, radial or vertical cell morphologies. Atypical neurons exhibited properties consistent with inhibitory interneurons, including tonic firing or initial bursting discharge, Ih currents, and islet cell morphology. Although both Typical and Atypical CR-expressing neurons responded to noxious peripheral stimulation, the excitatory drive onto Typical CR-expressing neurons was much stronger. Furthermore, Atypical CR-expressing cells comprise at least two functionally distinct subpopulations based on their responsiveness to noxious peripheral stimulation and neurochemical profile. Together our data suggest CR expression is not restricted to excitatory neurons in the SDH. Under normal conditions, the contribution of 'Typical' excitatory CR-expressing neurons to overall SDH excitability may be limited by the presence of A-type potassium currents, which limit the effectiveness of their strong excitatory input. Their contribution may, however, be increased in pathological situations where A-type potassium currents are decreased. By contrast, 'Atypical' inhibitory neurons with their excitable phenotype but weak excitatory input may be more easily recruited during increased peripheral stimulation.

HCN4 subunit expression in fast-spiking interneurons of the rat spinal cord and hippocampus.

Hyperpolarisation-activated (Ih) currents are considered important for dendritic integration, synaptic transmission, setting membrane potential and rhythmic action potential (AP) discharge in neurons of the central nervous system. Hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels underlie these currents and are composed of homo- and hetero-tetramers of HCN channel subunits (HCN1-4), which confer distinct biophysical properties on the channel. Despite understanding the structure-function relationships of HCN channels with different subunit stoichiometry, our knowledge of their expression in defined neuronal populations remains limited. Recently, we have shown that HCN subunit expression is a feature of a specific population of dorsal horn interneurons that exhibit high-frequency AP discharge. Here we expand on this observation and use neuroanatomical markers to first identify well-characterised neuronal populations in the lumbar spinal cord and hippocampus and subsequently determine whether HCN4 expression correlates with high-frequency AP discharge in these populations. In the spinal cord, HCN4 is expressed in several putative inhibitory interneuron populations including parvalbumin (PV)-expressing islet cells (84.1%; SD: ±2.87), in addition to all putative Renshaw cells and Ia inhibitory interneurons. Similarly, virtually all PV-expressing cells in the hippocampal CA1 subfield (93.5%; ±3.40) and the dentate gyrus (90.9%; ±6.38) also express HCN4. This HCN4 expression profile in inhibitory interneurons mirrors both the prevalence of Ih sub-threshold currents and high-frequency AP discharge. Our findings indicate that HCN4 subunits are expressed in several populations of spinal and hippocampal interneurons, which are known to express both Ih sub-threshold currents and exhibit high-frequency AP discharge. As HCN channel function plays a critical role in pain perception, learning and memory, and sleep as well as the pathogenesis of several neurological diseases, these findings provide important insights into the identity and neurochemical status of cells that could underlie such conditions.

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.

P boutons in lamina IX of the rodent spinal cord express high levels of glutamic acid decarboxylase-65 and originate from cells in deep medial dorsal horn.

Presynaptic inhibition of primary muscle spindle (group Ia) afferent terminals in motor nuclei of the spinal cord plays an important role in regulating motor output and is produced by a population of GABAergic axon terminals known as P boutons. Despite extensive investigation, the cells that mediate this control have not yet been identified. In this work, we use immunocytochemistry with confocal microscopy and EM to demonstrate that P boutons can be distinguished from other GABAergic terminals in the ventral horn of rat and mouse spinal cord by their high level of the glutamic acid decarboxylase (GAD) 65 isoform of GAD. By carrying out retrograde labeling from lamina IX in mice that express green fluorescent protein under the control of the GAD65 promoter, we provide evidence that the cells of origin of the P boutons are located in the medial part of laminae V and VI. Our results suggest that P boutons represent the major output of these cells within the ventral horn and are consistent with the view that presynaptic inhibition of proprioceptive afferents is mediated by specific populations of interneurons. They also provide a means of identifying P boutons that will be important in studies of the organization of presynaptic control of Ia afferents.

Selective loss of spinal GABAergic or glycinergic neurons is not necessary for development of thermal hyperalgesia in the chronic constriction injury model of neuropathic pain.

GABA and glycine are inhibitory neurotransmitters used by many neurons in the spinal dorsal horn, and intrathecal administration of GABA(A) and glycine receptor antagonists produces behavioural signs of allodynia, suggesting that these transmitters have an important role in spinal pain mechanisms. Several studies have described a substantial loss of GABA-immunoreactive neurons from the dorsal horn in nerve injury models, and it has been suggested that this may be associated with a loss of inhibition, which contributes to the behavioural signs of neuropathic pain. We have carried out a quantitative stereological analysis of the proportions of neurons in laminae I, II and III of the rat dorsal horn that show GABA- and/or glycine-immunoreactivity 2 weeks after nerve ligation in the chronic constriction injury (CCI) model, as well as in sham-operated and nai;ve animals. At this time, rats that had undergone CCI showed a significant reduction in the latency of withdrawal of the ipsilateral hindpaw to a radiant heat stimulus, suggesting that thermal hyperalgesia had developed. However, we did not observe any change in the proportion of neurons in laminae I-III of the ipsilateral dorsal horn that showed GABA- or glycine-immunoreactivity compared to the contralateral side in these animals, and these proportions did not differ significantly from those seen in sham-operated or nai;ve animals. In addition, we did not see any evidence for alterations of GABA- or glycine-immunostaining in the neuropil of laminae I-III in the animals that had undergone CCI. Our results suggest that significant loss of GABAergic or glycinergic neurons is not necessary for the development of thermal hyperalgesia in the CCI model of neuropathic pain.

Distribution and colocalisation of glutamate decarboxylase isoforms in the rat spinal cord.

The inhibitory neurotransmitter GABA is synthesized by glutamic acid decarboxylase (GAD), and two isoforms of this enzyme exist: GAD65 and GAD67. Immunocytochemical studies of the spinal cord have shown that whilst both are present in the dorsal horn, GAD67 is the predominant form in the ventral horn. The present study was carried out to determine the pattern of coexistence of the two GAD isoforms in axonal boutons in different laminae of the cord, and also to examine the relation of the GADs to the glycine transporter GLYT2 (a marker for glycinergic axons), since many spinal neurons are thought to use GABA and glycine as co-transmitters. Virtually all GAD-immunoreactive boutons throughout the spinal grey matter were labelled by both GAD65 and GAD67 antibodies; however, the relative intensity of staining with the two antibodies varied considerably. In the ventral horn, most immunoreactive boutons showed much stronger labelling with the GAD67 antibody, and many of these were also GLYT2 immunoreactive. However, clusters of boutons with high levels of GAD65 immunoreactivity were observed in the motor nuclei, and these were not labelled with the GLYT2 antibody. In the dorsal horn, some GAD-immunoreactive boutons had relatively high levels of labelling with either GAD65 or GAD67 antibody, whilst others showed a similar degree of labelling with both antibodies. GLYT2 immunoreactivity was associated with many GAD-immunoreactive boutons; however, this did not appear to be related to the pattern of GAD expression. It has recently been reported that there is selective depletion of GAD65, accompanied by a loss of GABAergic inhibition, in the ipsilateral dorsal horn in rats that have undergone peripheral nerve injuries [J Neurosci 22 (2002) 6724]. Our finding that some boutons in the superficial laminae showed relatively high levels of GAD65 and low levels of GAD67 immunoreactivity is therefore significant, since a reduction in GABA synthesis in these axons may contribute to neuropathic pain.

Modulation of inhibitory autapses and synapses on rat CA1 interneurones by GABA(A) receptor ligands.

To determine whether autaptic inhibition plays a functional role in the adult hippocampus, the action potential afterhyperpolarisations (spike AHPs) of CA1 interneurones were investigated in 25 basket, three bistratified and eight axo-axonic cells. The spike AHPs showed two minima in all regular-spiking (5), burst-firing (3) and in many fast-spiking cells (17:28). The fast component had a time-to-peak (TTP) of 1.2 +/- 0.5 ms, the slower TTP was very variable (range of 3.3-103 ms). The AHP width at half-amplitude (HW) was 12.5 +/- 5.7 ms in fast-spiking, 29.3 +/- 18 ms in regular-spiking and 99.7 +/- 42 ms in burst-firing cells. Axo-axonic cells never establish autapses, and the fast-spiking variety showed narrow (HW: 3.9 +/- 0.7 ms) spike AHPs with only one AHP minimum (TTP: 0.9 +/- 0.1 ms). When challenged with GABA(A) receptor modulators, spike AHPs in basket and bistratified cells were enhanced by zolpidem (HW by 18.4 +/- 6.2 % in 10:15 cells tested), diazepam (45.2 +/- 0.5 %, 6:7), etomidate (43.9 +/- 36 %, 6:8) and pentobarbitone sodium (41 %, 1:1), and were depressed by bicuculline (-41 +/- 5.7 %, 5:8) and picrotoxin (-54 %, 1:1), and the enhancement produced by zolpidem was reduced by flumazenil (-31 +/- 13 %, relative to the AHP HW during exposure to zolpidem, 3:4). Neuronal excitability was modulated in parallel. The spike AHPs of three axo-axonic cells tested showed no sensitivity to etomidate, pentobarbitone or diazepam. Interneurone-to-interneurone inhibitory postsynaptic potentials (IPSPs), studied with dual intracellular recordings, had time courses resembling those of the spike AHPs. The IPSP HW was 13.4 +/- 2.8 ms in fast-spiking (n = 16) and 28.7 +/- 5.8 ms in regular-spiking/burst-firing cells (n = 6), and the benzodiazepine1-selective modulator zolpidem strongly enhanced these IPSPs (45 +/- 28 %, n = 5). Interneurones with spike AHPs affected by the GABA(A) receptor ligands exhibited 3.8 +/- 1.9 close autaptic appositions. In three basket cells studied at the ultrastructural level 6 of 6, 1 of 2 and 1 of 2 close appositions were confirmed as autapses. Therefore, in the hippocampus autaptic connections contribute to spike AHPs in many interneurones. These autapses influence neuronal firing and responses to GABA(A) receptor ligands.

The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal horn.

Two vesicular glutamate transporters, VGLUT1 and VGLUT2, have recently been identified, and it has been reported that they are expressed by largely nonoverlapping populations of glutamatergic neurons in the brain. We have used immunocytochemistry with antibodies against both transporters, together with markers for various populations of spinal neurons, in an attempt to identify glutamatergic interneurons in the dorsal horn of the mid-lumbar spinal cord of the rat. The great majority (94-100%) of nonprimary axonal boutons that contained somatostatin, substance P or neurotensin, as well as 85% of those that contained enkephalin, were VGLUT2-immunoreactive, which suggests that most dorsal horn neurons that synthesize these peptides are glutamatergic. In support of this, we found that most somatostatin- and enkephalin-containing boutons (including somatostatin-immunoreactive boutons that lacked calcitonin gene-related peptide and were therefore probably derived from local interneurons) formed synapses at which AMPA receptors were present. We also investigated VGLUT expression in central terminals of primary afferents. Myelinated afferents were identified with cholera toxin B subunit; most of those in lamina I were VGLUT2-immunoreactive, whereas all those in deeper laminae were VGLUT1-immunoreactive, and some (in laminae III-VI) appeared to contain both transporters. However, peptidergic primary afferents that contained substance P or somatostatin (most of which are unmyelinated), as well as nonpeptidergic C fibres (identified with Bandeiraea simplicifolia isolectin B4) showed low levels of VGLUT2-immunoreactivity, or were not immunoreactive with either VGLUT antibody. As all primary afferents are thought to be glutamatergic, this raises the possibility that unmyelinated afferents, most of which are nociceptors, express a different vesicular glutamate transporter.

Gating of vagal inputs by sciatic afferents in nonspinally projecting neurons in the rat rostral ventrolateral medulla oblongata.

Integration and coordination of somato-visceral sensory information is crucial to achieve adaptive behavioural responses. We have recently shown that sensory vagal and somato-sensory (sciatic nerve) inputs converge in neurons of the rostral ventrolateral medulla oblongata, which was implicated in adjusting visceral activities to changing somatic performances. In the present study, the neuronal mechanism of interaction between sciatic and vagal sensory inputs was examined in the rostral ventrolateral medulla oblongata using in vivo intracellular recording and labelling. Conditioning stimulation of the contralateral sciatic nerve (2 V) led to a time-dependent inhibition of responses to vagal stimulation (100 microA) in each RVLM neuron that received convergent sciatic and vagal sensory inputs (n = 50). None of these neurons had direct spinal projections, and only 8% of them exhibited a visible response to stimulation of the aortic depressor nerve. A significant attenuation of the amplitude of vagal test responses was present for up to 800 ms of conditioning delay, although the duration of this sciatico-vagal inhibition was greatly dependent on the intensity of both stimuli. The electrophysiological data indicated that sciatico-vagal inhibition is mediated presynaptically, via activation of GABAB receptors. Morphological evidence of axo-axonic interactions that may underlie sciatico-vagal inhibition was subsequently found in the electron microscope. It is suggested that during movements of the hindleg, activation of sciatic sensory fibres leads to re-patterning of neuronal activity in RVLM neurons via inhibition of visceral sensory inputs. Sciatico-vagal inhibition is likely to affect the activity of those RVLM neurons that modulate higher neuronal activities via ascending projections.

Double immunofluorescence, peroxidase labelling and ultrastructural analysis of interneurones following prolonged electrophysiological recordings in vitro.

Inhibitory hippocampal and neocortical interneurones comprise a physiologically, morphologically and neurochemically heterogenous cell population. To identify the roles each class of interneurone plays within a given circuit it is necessary to correlate the electrophysiological properties of individual cells with their neurochemistry and morphology at both the light and electron microscopic level. However, the optimal conditions required for any one part of the protocol typically compromise the results from another. We have developed a protocol which allows the neurochemical content, gross morphology and ultrastructure details of biocytin-filled neurones to be recovered following long, dual intracellular recordings in thick mature slices maintained in an interface recording chamber, helping define sub-populations which could not otherwise be determined. Dual immunofluorescence is performed by incubating the tissue in monoclonal and polyclonal antibodies simultaneously, prior to visualization of biocytin-labelling with precipitation of a peroxidase reaction product. By using a biotinylated anti-avidin D antibody (Vector Laboratories), the intensity of this precipitation can be enhanced further where necessary. It is envisaged that this protocol can not only help determine the neurochemical content of cells recorded in similar in vivo studies, but that the ability to amplify peroxidase labelling in poorly filled cells is also of interest.

Differential sensitivity to Zolpidem of IPSPs activated by morphologically identified CA1 interneurons in slices of rat hippocampus.

Hippocampal pyramidal cells express several alpha-subunits, which determine the affinity of GABAA (gamma-aminobutyric acid) receptors for benzodiazepine site ligands. This study asked whether inhibitory postsynaptic potentials (IPSPs) elicited by specific interneuronal subclasses were differentially sensitive to the alpha1-preferring agonist Zolpidem, i.e. whether different receptors mediate different inhibitory connections. Paired intracellular recordings in which the presynaptic cell was an interneuron and the postsynaptic cell a CA1 pyramid were performed in slices of adult rat hippocampus. Resultant IPSPs were challenged with Zolpidem, cells filled with biocytin and identified morphologically. IPSPs elicited by fast spiking (FS) basket cells (n = 9) were enhanced more than IPSPs elicited by regular spiking (RS) basket cells (n = 10). At FS basket cell synapses the efficacy of Zolpidem was equivalent to that of Diazepam, while RS basket cell IPSPs are enhanced 50% less by Zolpidem than by Diazepam. Thus, while alpha1 subunits may dominate at synapses supplied by FS basket cells, RS basket cell synapses also involve alpha2/3 subunits. Two bistratified cell IPSPs tested with Zolpidem did not increase in amplitude, despite powerful enhancements of bistratified cell IPSPs by Diazepam, consistent with previous indications that these synapses utilize alpha5-containing receptors. Enhancements of basket cell IPSPs by Zolpidem and Diazepam were bi- or triphasic with steep amplitude increases separated by plateaux, occurring 10-15, 25-30 and 45-55 min after adding the drug to the bath. The entire enhancement was, however, blocked by the antagonist Flumazenil (n = 7). Flumazenil, either alone (n = 3), or after Zolpidem, reduced IPSP amplitude to approximately 90% of control, suggesting that alpha4-containing receptors were not involved.

Automatic attenuation compensation for ultrasonic imaging.

A new method has been developed for the automatic correction for attenuation in ultrasonic pulse-echo imaging. By assuming that a specified relationship holds between local attenuation and local backscatter throughout each pulse-echo signal, a gain factor may be calculated that more appropriately compensates for attenuation at every depth. The rationale depends on the observation that, broadly speaking, materials that backscatter strongly also attenuate strongly. The new method has been evaluated by postprocessing clinical B-scans following video frame capture. The images formed demonstrated the success of the algorithm in imaging homogeneous tissues at a constant grey level, irrespective of variations in the attenuation of overlying tissue. For example, the fetal lung was imaged uniformly irrespective of the presence of rib or spine, and prostatic tissue was imaged uniformly through a partial bladder window. Spatial resolution and speckle patterns are retained. The method is suitable for real-time application.