3D visualization and stereographic techniques for medical research and education.

While computers have been able to work with true 3D models for a long time, the same does not apply to the users in common. Over the years, a number of 3D visualization techniques have been developed to enable a scientist or a student, to see not only a flat representation of an object, but also an approximation of its Z-axis. In addition to the traditional flat image representation of a 3D object, at least four established methodologies exist: Stereo pairs. Using image analysis tools or 3D software, a set of images can be made, each representing the left and the right eye view of an object. Placed next to each other and viewed through a separator, the three dimensionality of an object can be perceived. While this is usually done on still images, tests at Mednet have shown this to work with interactively animated models as well. However, this technique requires some training and experience. Pseudo3D, such as VRML or QuickTime VR, where the interactive manipulation of a 3D model lets the user achieve a sense of the model's true proportions. While this technique works reasonably well, it is not a "true" stereographic visualization technique. Red/Green separation, i.e. "the traditional 3D image" where a red and a green representation of a model is superimposed at an angle corresponding to the viewing angle of the eyes and by using a similar set of eyeglasses, a person can create a mental 3D image. The end result does produce a sense of 3D but the effect is difficult to maintain. Alternating left/right eye systems. These systems (typified by the StereoGraphics CrystalEyes system) let the computer display a "left eye" image followed by a "right eye" image while simultaneously triggering the eyepiece to alternatively make one eye "blind". When run at 60 Hz or higher, the brain will fuse the left/right images together and the user will effectively see a 3D object. Depending on configurations, the alternating systems run at between 50 and 60 Hz, thereby creating a flickering effect, which is strenuous for prolonged use. However, all of the above have one or more drawbacks such as high costs, poor quality and localized use. A fifth system, recently released by Barco Systems, modifies the CrystalEyes system by projecting two superimposed images, using polarized light, with the wave plane of the left image at right angle to that of the right image. By using polarized glasses, each eye will see the appropriate image and true stereographic vision is achieved. While the system requires very expensive hardware, it solves some of the more important problems mentioned above, such as the capacity to use higher frame rates and the ability to display images to a large audience. Mednet has instigated a research project which uses reconstructed models from the central nervous system (human brain and basal ganglia, cortex, dendrites and dendritic spines) and peripheral nervous system (nodes of Ranvier and axoplasmic areas). The aim is to modify the models to fit the different visualization techniques mentioned above and compare a group of users perceived degree of 3D for each technique.

Haptic palpation of head and neck cancer patients--implication for education and telemedicine.

Malignancy in the head and neck area is a disease that often gives high morbidity in functions like speech, eating, breathing and cosmetics. To ensure a treatment of high clinical standard these patients are presented for a multidisciplinary tumor-team at Sahlgren University hospital. The team usually involves ENT-surgeons (Ear, Nose and Throat), oncologists, radiologists, pathologists, plastic surgeon, general surgeon and oral surgeons. The aim of the presentation is to classificate the tumor and suggests a treatment. The patients presented are from the whole western region of Sweden, and therefore some patients have to travel long distances. To minimize travel telemedicine was introduced 1998 with success [1]. One concern, when presenting a patient with telemedicine, has been the lack of possibility to palpate the tumor and the tissue surrounding it. To address this problem a 3D model of the tumor visualizes the region and possibly allows haptic palpation. Based on a series of high resolution CT/MR scans, a model of the region around the patients tumor is created. Haptic properties are added to the skin and subcutaneous structures (including the tumor) of the model. Initially, the haptic tuning is done by an examining physician, but in the final telemedical application, the aim is to develop a sensory device for this purpose (e.g. a position sensitive glove, such as Virtual Technologies, Inc. CyberGlove [2] and a graded system for setting firmness of the tissue). The model with its haptic properties can then be examined visually and haptically, the latter using a haptic device such as the SensAble PHANToM [3]. The present system uses a 3D model in VRML format based on reconstructed structures in the ROI (which includes the jawbones, the vertebra, the throat, major muscles and the skin) from high resolution CT. Haptic properties are added using MAGMA 2.5 (ReachIn Technologies AB, Sweden) [4]. Haptic force feedback is provided using a PHANToM Desktop (SensAble Technologies Inc) [3]. Visual feedback can be either monoscopic or stereoscopic (StereoGraphic CrystalEyes) [5]. The system will be used for concept testing and for evaluating possible limitations and/or the need for a modified examination protocol. Once a reliable set of parameters has been generated (using both professionals and medical students at various levels), the remote components will be added.

Modeling and modification of medical 3D objects. The benefit of using a haptic modeling tool.

The Computer Laboratory of the medical faculty in Goteborg (Mednet) has since the end of 1998 been one of a limited numbers of participants in the development of a new modeling tool together with SensAble Technologies Inc [http:¿www.sensable.com/]. The software called SensAble FreeForm was officially released at Siggraph September 1999. Briefly, the software mimics the modeling techniques traditionally used by clay artists. An imported model or a user defined block of "clay" can be modified using different tools such as a ball, square block, scrape etc via the use of a SensAble Technologies PHANToM haptic arm. The model will deform in 3D as a result of touching the "clay" with any selected tool and the amount of deformation is linear to the force applied. By getting instantaneous haptic as well as visual feedback, precise and intuitive changes are easily made. While SensAble FreeForm lacks several of the features normally associated with a 3D modeling program (such as text handling, application of surface and bumpmaps, high-end rendering engines, etc) it's strength lies in the ability to rapidly create non-geometric 3D models. For medical use, very few anatomically correct models are created from scratch. However, FreeForm features tools enable advanced modification of reconstructed or 3D scanned models. One of the main problems with 3D laserscanning of medical specimens is that the technique usually leaves holes or gaps in the dataset corresponding to areas in shadows such as orifices, deep grooves etc. By using FreeForms different tools, these defects are easily corrected and gaps are filled out. Similarly, traditional 3D reconstruction (based on serial sections etc) often shows artifacts as a result of the triangulation and/or tessellation processes. These artifacts usually manifest as unnatural ridges or uneven areas ("the accordion effect"). FreeForm contains a smoothing algorithm that enables the user to select an area to be modified and subsequently apply any given amount of smoothing to the object. While the final objects need to be exported for further 3D graphic manipulation, FreeForm addresses one of the most time consuming problems of 3D modeling: modification and creation of non-geometric 3D objects.

Virtual reality on the web: the potentials of different methodologies and visualization techniques for scientific research and medical education.

Academic and medical imaging are increasingly using computer based 3D reconstruction and/or visualization. Three-dimensional interactive models play a major role in areas such as preclinical medical education, clinical visualization and medical research. While 3D is comparably easy to do on a high end workstations, distribution and use of interactive 3D graphics necessitate the use of personal computers and the web. Several new techniques have been demonstrated providing interactive 3D via a web browser thereby allowing a limited version of VR to be experienced by a larger majority of students, medical practitioners and researchers. These techniques include QuickTimeVR2 (QTVR), VRML2, QuickDraw3D, OpenGL and Java3D. In order to test the usability of the different techniques, Mednet have initiated a number of projects designed to evaluate the potentials of 3D techniques for scientific reporting, clinical visualization and medical education. These include datasets created by manual tracing followed by triangulation, smoothing and 3D visualization, MRI or high-resolution laserscanning. Preliminary results indicate that both VRML and QTVR fulfills most of the requirements of web based, interactive 3D visualization, whereas QuickDraw3D is too limited. Presently, the JAVA 3D has not yet reached a level where in depth testing is possible. The use of high-resolution laserscanning is an important addition to 3D digitization.

Laser 3-D scanning for surface rendering in biomedical research and education.

A technique based on laser scanning is applied to body parts and organs. Laser light is projected onto the surface of objects and recorded by CCD sensors. This is a fast and flexible method for accurately scanning surface geometry. It also allows conversion to NURBS patches. We have, so far, made 'point-cloud' surface renderings from a head model, a plastic brain model, a human brain, moulds of bites and a cranium. The limits, quality, efforts and costs of employing the laser 3-D scanning technique are evaluated. The experiments are currently in progress and results give interesting 3D renderings and attempts to triangulated solid-models.

3D-brain 2.0--narrowing the gap between personal computers and high end workstations.

UNLABELLED: Recent advances in personal computer hardware and software have pushed the graphic capacity of these easier to use and, more importantly, cheaper computers to a level approximating the current standard of high end workstations. The interactivity and graphic complexity of a modern PC is rapidly approaching the current standard on Silicon Graphics (although with respect to texture mapping, the SGI is still ahead of the PCs). The modern medical student laboring under increasingly higher demands with respect to versatility, not only in basic science and traditional medical knowledge, is also faced with the requirement to learn and understand modern scientific visualization and analytical instruments. Furthermore, basic knowledge of information technology and computer literacy is expected of the next generation medical professionals. These demands forces medical schools to increasingly invest in computers and information technology for educational purposes. Due to common class sizes, these computers are most commonly Windows PCs or Apple Macintoshes. For distance education, telematics or studies at home, personal computer versions of the workstation graphics are a necessity. 3D-Brain 2.0 is an educational software package intended to run on basic personal computers and utilizing modern software technologies such as QuickTime VR 2.0 and VRML 2.0, to provide the students with insight into modern clinical and scientific visualization, focusing on the anatomy and functionality of the human brain. The aim of this paper to test the validity and usefulness of these new visualization techniques. METHODS: 3D-Brain is based on human brains sliced in 1 mm sections (NB. NOT based on NLMs Visual Human). Each slice was photographed, digitized, optimized and aligned using proprietary software. The datasets were then created by manual tracing followed by triangulation, smoothing and 3D visualization using Silicon Graphics computers. For the QuickTime VR project, 684 images with a 10 degrees angle were generated for each scene and ported to an Apple Macintosh computer for further manipulation. VRML code was generated directly from the original dataset. All interactivity was programmed on a Macintosh and subsequently ported to the Windows95 PC platform. The minimum requirements to run the software are either a PowerPC based Macintosh computer or a Pentium based Windows 95 computer with 16 Mb, 16 bit display and a 4 speed CD-ROM. RESULTS AND DISCUSSION: 3D-Brain 2.0 provides medical students at Goteborg University the means to complement traditional teaching using visualization techniques and three-dimensional models. These techniques also serve as an insight into the different clinical means of visualization the student will encounter throughout his/her continued education and professional career. For educational purposes, it has been established that among the tested new visualization techniques, CD-ROM based software utilizing QTVR is still the best methodology to use for pedagogical software. VRML shows promise in porting these software packages to the Web while Open Inventor is the preferred format for research purposes.

The BRAIN project: an interactive learning tool using desktop virtual reality on personal computers.

The BRAIN-project is an endeavor in using computer aided learning to improve the understanding of the human brain anatomy. The project consists of four parts, each based on modular packages: BRAINIMAGES: Brain atlas consisting of horizontal and frontal brain slices spaced I mm apart enabling the identification of structures and areas of the brain. The software also contains views of the brain's outer surface with all pertinent structures marked. BRAINRADIOLOGY: Visualisation of the brain using CT, MRI and angiography. The different imaging techniques enable the user to explore the brain from several angles and also view the major blood vessels of the brain. NEUROHISTOLOGY: Cells of the brain using histologically stained sections. The program emphasizes the organization of cells in layers and the interaction of different cell types. 3D-BRAIN: Three dimensional reconstructions based on physical slices of a human brain. The reconstructed brain views are made interactive using a simple form of desktop virtual reality: QuickTime VR technology. The user can rotate the different views in all directions producing a 3D effect. The different views are designed to highlight important structures and their organization within the outlined (and semi transparent) brain surface. Contrary to similar applications, the actual three dimensional objects are not based on MRI or CT scans (with comparatively poor resolution), but on tracings made on high resolution images of photographs of actual sections of a postmortem brain. N.B., this approach produces 3D renderings in a more detailed and reliable way. The BRAIN project is designed as a support package for students in preclinical education by supplying additional means for gathering information pertinent to the curriculum. By cross linking, the students can switch from a three dimensional object to a corresponding slice, and then to the relevant histological sample and son on. The software components are based on a modular design enabling easy modification of the various parts and the entire project is designed to run on both Apple Macintosh and MS Windows based PCs.

Effects on locomotor activity after local application of (+)-UH232 in discrete areas of the rat brain.

The preferential dopamine autoreceptor, and slightly D3 preferring, antagonist (+)-UH232 (cis-(+)-(1S,2R)-5-methoxy-1-methyl-2-(n-dipropylamino) tetralin) increases locomotor activity and synaptic dopamine release in the nucleus accumbens and striatum after systemic administration to the rat. As shown in this study, (+)-UH232, was unable to produce an increase in locomotor activity measured for 60 minutes after local administration into the terminal or somato-dendritic regions of the mesolimbic dopamine pathways or into the lateral ventricle. Instead, a dose dependent decrease of spontaneous locomotor activity after local application (0.05-50.0 nmol/side) of (+)-UH232 into the nucleus accumbens, was seen. A similar reduction in locomotor activity was produced by the classical dopamine antagonist raclopride. Analysis of the dose*time interactions on locomotor activity did, however, indicate that there is a significant dose*time interaction after local application of (+)-UH232 into the lateral ventricle and VTA. Raclopride, on the other hand, produced only a weak time dependent effect in the VTA. The potential problem of Leao's spreading depression in micro-injection experiments were considered, however, spreading depression does not seem to influence the effects of (+)-UH232 locally applied into the nucleus accumbens. In conclusion, both (+)-UH232 and raclopride produced a dose dependent decrease in spontaneous locomotor activity when examined as the total activity count over 60 minutes after local application into the N Acc.

Effects of the dopamine D3-and autoreceptor preferring antagonist (-)-DS121 on locomotor activity, conditioned place preference and intracranial self-stimulation in the rat.

The phenylpiperidine (-)-DS121 (S-(-)-3-(-3-cyanophenyl)-N-n-propyl piperidine) represents a new class of weak stimulants acting as preferential dopamine autoreceptor antagonists. (-)-DS121 dose-dependently increases locomotor activity over a wide dose range in rats after systemic administration. (-)-DS121 also exhibits a weak preference for the D3 receptor in in vitro binding studies. The relevance of this D3 preference is not clear and it is not known whether the D3 receptor site influences reward mechanisms. The present results showed that (-)-DS121 induced place conditioning in the dose range 3.3-13.3mg/kg s.c. as did d-amphetamine (0.25-4.0mg/kg, s.c.). However, in contrast to d-amphetamine, (-)-DS121 failed to facilitate infracranial self-stimulation in the dose range that produced place conditioning. Local bilateral infusion of (-)-DS121 (0.05-53.0µg/side) into the nucleus accumbens or ventral tegmental area did not produce locomotor stimulation. A weak but significant increase in locomotor activity was detected after bilateral infusion of (-)-DS121 (66.3µg/side) into the lateral ventricles. This study suggests that the behavioural stimulant (-)-DS121 does not possess strong reward-facilitating properties and that local application in either the terminal or somatodendritic regions of the mesolimbic pathway does not produce the same degree of locomotor activity as seen after systemic administration.

Effects of dopamine D3 preferring compounds on conditioned place preference and intracranial self-stimulation in the rat.

Compounds showing an in vitro binding preference for the dopamine D3 receptor were tested in two models designed to assess positive reinforcement in the rat: intracranial self-stimulation (ICSS) and conditioned place preference (CPP). R-(+)-7-OH-DPAT, a D3 preferring agonist, inhibited ICSS behaviour over a wide dose range. At higher doses, a facilitation of ICSS was seen. In the CPP model, 7-OH-DPAT was inactive except at the highest dose where a significant change in preference was seen. A dose of R-(+)-7-OH-DPAT, that significantly inhibited ICSS behaviour, was combined with a dose of d-amphetamine, that significantly facilitated ICSS behaviour. Surprisingly, this resulted in a significant synergistic facilitation of the amphetamine response. The putative D3 antagonist, U99194A was inactive in the ICSS model but induced significant place preference. The present results suggest that the dopamine D3 receptor, in contrast to the D2 receptor, has an inhibitory influence on reward mechanisms.

Effects on locomotor activity after local application of D3 preferring compounds in discrete areas of the rat brain.

Compounds showing an in vitro binding preference for the dopamine D3 vs. D2 receptors were tested for effects on locomotor activity after local application in the nucleus accumbens (N Acc) and the ventral tegmental area (VTA) of the rat brain. R-(+)-7-OH-DPAT, a dopamine D3 preferring agonist, inhibited spontaneous locomotor activity over a wide dose range after injection into the N Acc. A decrease in activity over a wide dose range was also seen after local application into the VTA of both R-(+)-7-OH-DPAT and the dopamine D2 preferring agonist (+)-3-PPP. Furthermore, (+)-3-PPP produced a dose dependent increase in activity after local application into the N Acc. The putative D3 antagonist, U99194A, with a 30 fold preference for the dopamine D3 vs. D2 receptor, produced an increase in activity when injected into the N Acc. A similar pattern were seen after infusion into the lateral ventricle. Local application into the VTA did, however, not produce any significant effects. The present results support the hypothesis that dopamine D3 receptors (in contrast to the D2 receptors) are mainly postsynaptically located where they display an inhibitory action on locomotor activity.

Behavioral and neurochemical data suggest functional differences between dopamine D2 and D3 receptors.

In an in vitro model for mitogenic activity in cloned Chinese hamster ovary (CHO) cells expressing rat dopamine D2 or D3 receptors, the EC50D2/EC50D3 ratios for the agonists, apomorphine, (+)-3-hydroxy-N-n-propyl-phenylpiperidine ((+)-3-PPP), quinpirole, R-(+)-7-hydroxy-2-(di-n-propylamino)tetralin (R-(+)-7-OH-DPAT) and pramipexole (SND919) were found to be 0.36, 0.41, 1.3, 3.7 and 7.0, respectively. In locomotor activity experiments with actively exploring rats, the more dopamine D3 preferring agonists, R-(+)-7-OH-DPAT and pramipexole, were most efficacious to reduce locomotion. The hypoactivity was also observed at doses that did not affect brain dopamine synthesis rate (DOPA accumulation) or release (measured in in vivo dialysis experiments). In contrast, for apomorphine, (+)-3-PPP and quinpirole there was a closer correlation between doses that reduced exploratory activity and doses that reduced brain dopamine release and synthesis. The present data support the hypothesis that the functional dopamine D3 receptor is a postsynaptic receptor inhibitory on rat locomotion.

The preferential dopamine autoreceptor antagonist (+)-UH232 antagonizes the positive reinforcing effects of cocaine and d-amphetamine in the ICSS paradigm.

The dopamine autoreceptor and D3 preferring antagonist [cis-(+)-5-methoxy-1-methyl-2-(di-n-propylamino)tetralin] (+)-UH232, exerts weak stimulatory effects when tested in locomotor activity experiments using habituated animals. (+)-UH232 also blocks d-amphetamine-, cocaine-, and apomorphine-induced hyperactivity, but fails to induce catalepsy. Thus, the behavioral effects of (+)-UH232 appear to be dependent upon the baseline activity of the animal. The antagonistic properties of (+)-UH232 were studied in the intracranial self-stimulation (ICSS) technique in the rat. (+)-UH232 and haloperidol produced inhibitory effects over a wide dose range. Cocaine, GBR12909 and d-amphetamine clearly lowered ICSS thresholds, indicating stimulatory effects. (+)-UH232 antagonized the stimulatory effects of cocaine, GBR12909, and d-amphetamine, whereas haloperidol, at a dose producing an inhibition similar to (+)-UH232, was significantly weaker in antagonizing cocaine- or d-amphetamine-induced stimulation. This difference between (+)-UH232 and haloperidol with respect to stimulant-blocking ability, support the concept that the effects of (+)-UH232 are not representative of either classical DA agonists or DA antagonists.

GABAB-receptor activation alters the firing pattern of dopamine neurons in the rat substantia nigra.

Previous electrophysiological experiments have emphasized the importance of the firing pattern for the functioning of midbrain dopamine (DA) neurons. In this regard, excitatory amino acid receptors appear to constitute an important modulatory control mechanism. In the present study, extracellular recording techniques were used to investigate the significance of GABAB-receptor activation for the firing properties of DA neurons in the substantia nigra (SN) in the rat. Intravenous administration of the GABAB-receptor agonist baclofen (1-16 mg/kg) was associated with a dose-dependent regularization of the firing pattern, concomitant with a reduction in burst firing. At higher doses (16-32 mg/kg), the firing rate of the DA neurons was dose-dependently decreased. Also, microiontophoretic application of baclofen regularized the firing pattern of nigral DA neurons, including a reduction of burst firing. Both the regularization of the firing pattern and inhibition of firing rate produced by systemic baclofen administration was antagonized by the GABAB-receptor antagonist CGP 35348 (200 mg/kg, i.v.). The GABAA-receptor agonist muscimol produced effects on the firing properties of DA neurons that were opposite to those observed following baclofen, i.e., an increase in firing rate accompanied by a decreased regularity. The NMDA receptor antagonist MK 801 (0.4-3.2 mg/kg, i.v.) produced a moderate, dose-dependent increase in the firing rate of the nigral DA neurons as well as a slightly regularized firing pattern. Pretreatment with MK 801 (3.2 mg/kg, i.v., 3-10 min) did neither promote nor prevent the regularization of the firing pattern or inhibition of firing rate on the nigral DA neurons produced by baclofen. The present results clearly show that GABAB-receptors can alter the firing pattern of nigral DA neurons, hereby counterbalancing the previously described ability of glutamate to induce burst firing activity on these neurons.

A simple computer-based method for performing and analyzing intracranial self-stimulation experiments in rats.

Intracranial self-stimulation (ICSS) in the rat is a useful tool for studying the importance of various brain monoamines in positive reinforcement. The effects of compounds interacting with dopaminergic neurotransmission is measurable by studying the changes of reward thresholds. By computerisation of the analysis of these thresholds, standardisation and reproducibility is greatly enhanced. The use of an object-oriented programming language simplifies the programming of a specific application and it provides scientists without formal training in computer programming the means to create their own software. A system for the acquisition, execution, analysis and storage of ICSS experiments is described. The hardware is based on Apple Macintosh computers, interfaced to the test chambers and physiological stimulators using a plug-in card supporting A/D, D/A, digital I/O and timer functions. The software written in G (LabVIEW) provides the user with a graphically based 'Virtual Instrument' performing all aspect of the ICSS experiment. The software performs threshold analysis immediately after completion of the ICSS experiment, thereby greatly reducing the total time previously needed to evaluate these experiments. The graphical approach used in LabVIEW allows the programmer to make fast and simple alterations to suit different experimental problems.

Effects of the preferential dopamine autoreceptor antagonist (+)-AJ76 in the intracranial self-stimulation paradigm.

As revealed by locomotor activity experiments in rodents, cis-(1S,2R)-5-methoxy-1-methyl-(2-n-propylamino)tetralin [(+)-AJ76] is a preferential dopamine autoreceptor antagonist that produces stimulatory or weak inhibitory behavioral effects in animals that display low or high baseline activity, respectively. In the present study, the possible positive reinforcing properties of (+)-AJ76 were studied by means of the intracranial (median forebrain bundle) self-stimulation (ICSS) technique in rats. The current intensity of the electrical stimuli was used as the independent variable. The resulting rate/intensity curves were analyzed by computer, and the half-maximal response (called EC50) was calculated for each animal. When starting on a suprathreshold current intensity, (+)-AJ76 dose dependently (3.1-52.0 microM/kg, SC) increased the EC50 without producing any apparent motor deficits like muscular rigidity or catalepsy. A clear-cut and more potent inhibitory action was also noted for haloperidol (0.033-0.133 microM/kg, SC) and the di-N-methyl analog of (+)-AJ76 called (+)-AJ118 (0.8-3.5 microM/kg, SC), while d-amphetamine (1.4 or 5.4 microM/kg, SC) decreased the EC50 values. In the second experiment, animals were subjected to a subthreshold current intensity for 30 min. The intensity was set to produce a response of 15% or less of maximal, shaping response rate for the respective animals. Of these 22 animals, 10 responded with a stimulation, while the ICSS response was inhibited in the others. We did not, however, get consistent results in all rats tested. In summary, this study shows that (+)-AJ76 appears to lack positive reinforcing properties comparable to those produced by classical stimulants such as d-amphetamine.

Quantification of axonally transported material using cytofluorimetric scanning.

This paper describes in detail a cytofluorimetric scanning technique used for studying amounts of material axonally transported in antero- and retrograde direction in peripheral nerves. Operating procedures, preparation of tissues and instrumental set-up are described. The basis for quantification of material in a nerve section treated for immunofluorescence is discussed. The reliability of the method has been tested by comparing results with biochemical data. There are several advantages of the technique. (1) Many different substances can be studied in one single nerve segment, thus reducing biological variation and costs. (2) Both morphological data and quantitative figures can be obtained; following scanning the section can be photographed. (3) The method can also be used on studies in the central nervous system and on tissue cultures, since it is possible to scan on single axons or bundles of fibres.

Serotonin-producing carcinoid tumour cells in long-term culture. Studies on serotonin release and morphological features.

Tumour cells from a hepatic metastasis of a midgut carcinoid tumour were studied during 240 days of culture. A cell line could not be established, but the cells regularly formed large clusters and islets. The spontaneous release of serotonin (5-HT) and neuropeptide K-like immunoreactivity from cultures were followed. The amine and the peptide levels were unstable without evident covariation. The response to stimulation with noradrenaline and isoprenaline was studied during the culture period. The tumour cells released 5-HT selectively at stimulation with isoprenaline. This responsiveness also showed considerable variation with long periods of quiescence. Ultrastructurally the tumour cells showed a certain degree of polarization with apical microvilli and a supranuclear Golgi apparatus. When studied by confocal laser scanning the tumour cells were demonstrated to be cylindrical in shape with a cytoplasmic attachment to the matrix.