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This is a summary of the 2022 Nansen Neuroscience Lectures. On 10 October 2022, Professors Henriette van Praag and David Gems gave the 2022 Nansen Neuroscience Lectures on the theme “Is ageing inevitable?” in the Norwegian Academy of Science and Letters, Oslo, Norway. While van Praag gave a lecture entitled “The benefits of exercise for brain function”, Gems gave the 2ndlecture discussing “What causes ageing? Lessons from The Worm”. Understanding the fundamental mechanisms of ageing will pave the way to the development of future interventions to pre-empt the development of the diseases, including Alzheimer’s disease and other dementias, of later life.

An increasing number of imaging techniques are in use to study the localization of molecules involved in cell-to-cell signaling. Here we describe the use of immunogold procedures to detect and quantify molecules on electron micrographs. To measure the areas of the subcellular compartments under investigation, the protocol uses an overlay screen with an array of regularly spaced points. On the basis of this, the densities of the gold-labeled molecules can be calculated. Despite the limited lateral resolution of the immunogold method as used by many investigators (∼30 nm), it is possible to measure the content of molecules associated with tiny tissue compartments, e.g., synaptic vesicles and different types of membrane, such as plasma membranes and vesicle membranes. The quantification protocol can be carried out without using computer programs. The entire protocol can be completed in ∼15 d.

The adipocyte does not only serve as fuel storage but produces and secretes compounds with modulating effects on food intake and energy homeostasis. Although there is firm evidence for a centrally mediated regulation of adipocyte function via the autonomous nervous system, little is known about signaling between adipocytes. Amino acid neurotransmitters are candidates for such paracrine signaling. Here, we applied immunohistochemistry to detect components required for amino acid transmitter signaling in rat fat depots. In interscapular brown adipose tissue as well as in interscapular, mesenteric, perirenal, and epididymal white adipose tissues, we demonstrate robust immunosignals for the excitatory neurotransmitter glutamate, the inhibitory neurotransmitter γ-aminobutyric acid (GABA), and the GABA-synthesizing enzyme glutamate decarboxylase (GAD) isoforms GAD65 and GAD67. Moreover, all adipose tissues stained for the vesicular glutamate transporter VGLUT1 and the vesicular GABA transporter VGAT in addition to the vesicle marker synaptophysin. Electron microscopic immunocytochemistry showed that VGLUT1 and VGAT, but not VGLUT2 or VGLUT3, are localized in vesicular organelles in adipocytes. The receptors for glutamate (subunits GluR2/3 and NR1 but not mGluR2) and for GABA (GABAARα2) were present in the adipocytes. The presence of glutamate, GABA, their vesicular transporters, and their receptors indicates a paracrine signaling role for amino acids in adipose tissues.

The adipocyte does not only serve as fuel storage but produces and secretes compounds with modulating effects on food intake and energy homeostasis. Although there is firm evidence for a centrally mediated regulation of adipocyte function via the autonomous nervous system, little is known about signaling between adipocytes. Amino acid neurotransmitters are candidates for such paracrine signaling. Here, we applied immunohistochemistry to detect components required for amino acid transmitter signaling in rat fat depots. In interscapular brown adipose tissue as well as in interscapular, mesenteric, perirenal, and epididymal white adipose tissues, we demonstrate robust immunosignals for the excitatory neurotransmitter glutamate, the inhibitory neurotransmitter γ-aminobutyric acid (GABA), and the GABA-synthesizing enzyme glutamate decarboxylase (GAD) isoforms GAD65 and GAD67. Moreover, all adipose tissues stained for the vesicular glutamate transporter VGLUT1 and the vesicular GABA transporter VGAT in addition to the vesicle marker synaptophysin. Electron microscopic immunocytochemistry showed that VGLUT1 and VGAT, but not VGLUT2 or VGLUT3, are localized in vesicular organelles in adipocytes. The receptors for glutamate (subunits GluR2/3 and NR1 but not mGluR2) and for GABA (GABAARα2) were present in the adipocytes. The presence of glutamate, GABA, their vesicular transporters, and their receptors indicates a paracrine signaling role for amino acids in adipose tissues.

Postembedding immunocytochemistry was used to localize aspartate, glutamate, gamma-aminobutyric acid (GABA), and glutamine in hippocampus and striatum during normo- and hypoglycemia in rat. In both brain regions, hypoglycemia caused aspartatelike immunoreactivity to increase. In hippocampus, this increase was evident particularly in the terminals of known excitatory afferents—in GABA-ergic neurons and myelinated axons. Aspartate was enriched along with glutamate in nerve terminals forming asymmetric synapses on spines and with GABA in terminals forming symmetric synapses on granule and pyramidal cell bodies. In both types of terminal, aspartate was associated with clusters of synaptic vesicles. Glutamate and glutamine immunolabeling were markedly reduced in all tissue elements in both brain regions, but less in the terminals than in the dendrosomatic compartments of excitatory neurons. In glial cells, glutamine labeling showed only slight attenuation. The level of GABA immunolabeling did not change significantly during hypoglycemia. The results support the view that glutamate and glutamine are used as energy substrates in hypoglycemia. Under these conditions both excitatory and inhibitory terminals are enriched with aspartate, which may be released from these nerve endings and thus contribute to the pattern of neuronal death characteristic of hypoglycemia.

After axotomy in the ventral funiculus of the cat spinal cord, about half of the population of lesioned motoneurons die at 1–3 weeks postoperatively, whereas the other half survives and generates new axons through the lesion area. To identify conditions that may promote survival and regeneration of motoneurons subjected to this kind of injury, the authors examined ultrastructurally lesion‐induced changes in the number and distribution of nerve terminals on the somata and proximal dendrites of α‐motoneurons in the 7th lumbar spinal segment (L7) of the cat spinal cord. Intramedullary axotomy resulted in a profound reduction in the number of nerve terminals impinging on the somata and proximal dendrites, with the maximal effect seen at 3 weeks postlesion. At that time, only 12–25% of the normal number of terminals remained on the cell somata, and 22–33% remained on proximal dendrites. Thereafter, a gradual increase in terminal numbers occurred, reaching normal levels at 34 weeks after the lesion. Already at 2 days postoperatively and, most obviously, at 3 weeks postoperatively, type S nerve terminals were eliminated to a larger degree than type F terminals. Postembedding immunohistochemistry confirmed that the largest reduction at 3 weeks was seen for excitatory glutamate‐immunopositive type S nerve terminals (90%), whereas inhibitory glycine‐immunoreactive and γ‐aminobutyric acid (GABA)‐immunoreactive type F terminals were affected less (70% reduction). This led to a distinct shift in the ratio between the numbers of terminals that were immunopositive for glycine and GABA and the numbers of terminals that were labeled for glutamate. For the cell body, this ratio increased from 3.7 in normal material to 14.5 in lesioned motoneurons, whereas the corresponding values for proximal dendrites were 3.8 and 7.5. The preferential elimination of glutamatergic inputs to lesioned motoneurons may reflect an active reorganization of the synaptic input to diminish the excitotoxic influence on these neurons, thereby promoting the survival of motoneurons after intramedullary axotomy. J. Comp. Neurol. 425:10–23, 2000. © 2000 Wiley‐Liss, Inc.

There is general agreement that last-order premotor interneurons—a set of neurons that integrate activities generated by the spinal motor apparatus, sensory information and volleys arising from higher motor centres, and transmit the integrated signals to motoneurons through monosynaptic contacts—play crucial roles in the initiation and maintenance of spinal motor activities. Here, we demonstrate the development, neurochemical properties, and axonal projections of a unique group of last-order premotor interneurons within the ventrolateral aspect of the lateral funiculus of the chick lumbosacral spinal cord. Neurons expressing immunoreactivity for neuron-specific enolase were first detected in the ventrolateral white matter at embryonic day 9 (E9). The numbers of immunoreactive neurons were significantly increased at E10–E12, while most of them were gradually concentrated in small segmentally arranged nuclei (referred to as major nuclei of Hofmann) protruding from the white matter in a necklace like fashion dorsal to the ventral roots. The major nuclei of Hofmann became more prominent at E12–E16, but substantial numbers of cells were still located within the ventrolateral white matter (referred to as minor nucleus of Hofmann). The distribution of immunoreactive neurons achieved by E16 was maintained during later developmental stages and was also characteristic of adult animals. After injection of Phaseolus vulgaris-leucoagglutinin unilaterally into the minor nucleus of Hofmann, labeled fibres were detected in the ventrolateral white matter ipsilateral to the injection site. Ascending and descending fibres were revealed throughout the entire rostro-caudal length of the lumbosacral spinal cord. Axon terminals were predominantly found within the lateral motor column and the ventral regions of lamina VII ipsilateral to the injection site. Several axon varicosities made close appositions with somata and dendrites of motoneurons, which were identified as synaptic contacts in a consecutive electron microscopic study. With the postembedding immunogold method, 21 of 97 labeled terminals investigated were immunoreactive for glycine and 2 of them showed immunoreactivity for γ-aminobutyric acid (GABA). The axon trajectories of neurons within the minor nucleus of Hofmann suggest that some of these cells might represent a population of last-order premotor interneurons. J. Exp. Zool. 286:157–172, 2000. © 2000 Wiley-Liss, Inc.

The monosynaptic connection between muscle spindle Ia afferents and motoneurones is one of the most extensively studied reflex pathways in the central nervous system. Physiological evidence has pointed to glutamate as the fast transmitter in this synaptic connection. In this study of the spinal cord segment C2, we used dual labelling with cholera toxin subunit B-horseradish peroxidase (B-HRP) and postembedding immunohistochemistry to show that group Ia primary afferent boutons synapsing on retrogradely labelled central cervical nucleus (CCN) neurones and motoneurones are enriched in glutamate-like immunoreactivity (LI). All double-labelled Ia terminals studied could be classified as S-type (CCN neurones and motoneurones) or M-type (motoneurones) boutons.

Summary The cellular and subcellular localization of glutamine, a major glutamate precursor, was studied by means of an antiserum raised against glutaraldehydefixed glutamine. Ultrathin sections from the cerebellar cortex of rat and baboon (Papio anubis) were incubated sequentially in the primary antiserum and in a secondary antibody coupled to colloidal gold particles. The labelling intensity was quantified by computer-aided calculation of gold particle densities. High levels of immunoreactivity occurred in glial cells (Bergmann fibres, astrocytes, and oligodendrocytes), intermediate levels in cell bodies and processes of granule cells, and low levels in terminals of presumed GABAergic or glutamatergic fibres (terminals of basket and Golgi cells, and of parallel, mossy, and climbing fibres). The labelling intensity of Purkinje cells showed some variation, but never exceeded that in glial cells. Within the nerve fibre terminals, the glutamine-like immunoreactivity showed some preference for mitochondria, but was otherwise evenly distributed. The predominant glial localization of glutamine was also obvious in light microscopic preparations processed according to the postembedding peroxidase-antiperoxidase procedure. Gold particle densities over different types of profile in glutamine immunolabelled sections were compared with particle densities over the corresponding types of profiles in neighbouring sections labelled with an antiserum to glutaraldehyde-fixed glutamate. The glutamate/glutamine ratio, expressed arbitrarily by the ratio between the respective gold particle densities, varied by a factor of about 6, with the highest ratio in the putative glutamatergic mossy and parallel fibre terminals, and the lowest ratio in glial elements. The remaining tissue components displayed intermediate ratios. The present study provides direct morphological evidence for the existence in the brain of distinct compartments with differing glutamate/glutamine ratios.

Frozen sections of the corpus ventriculi, antrum pyloricum, duodenum, jejunum, ileum and colon from animals perfusion fixed with glutaraldehyde were treated with an antiserum specific for glutaraldehyde-fixed GABA and processed by the peroxidase antiperoxidase method. Semithin plastic sections from the antrum pyloricum were treated similarly. Stained cells appeared in the epithelium of all segments examined except the corpus ventriculi. The highest density of cells was observed along the major curvature of the antrum pyloricum. Here they were located in the bottom half of the gastric glands. Many of the cells showed a process extending towards the glandular lumen. No significant staining in the epithelium appeared when the antiserum was preincubated with glutaraldehyde-GABA complexes, nor when the anti-GABA serum was exchanged with anti-glycine or preimmune serum. The present findings and previous physiological data suggest that GABA may play a role in gut endocrine regulation.

Antisera produced by immunizing rabbits with GABA conjugated to bovine serum albumin reacted, after purification, strongly with GABA fixed with glutaraldehyde to rat brain macromolecules, but insignificantly with other fixed amino acids (Storm-Mathisen et al. 1983). Sections through the diencephalon of perfusion-fixed mouse and rat brains showed a highly selective labeling pattern after incubation with these antisera. All cells of the reticular nucleus appeared to be stained. Smaller proportions of stained perikarya occurred in the dorsal and ventral subdivisions of the lateral geniculate body, in the medial geniculate body, in the lateroposterior nucleus, and in all nuclei of the pretectum. Labeled cell bodies were only rarely encountered in the ventrobasal complex, and were not found in the anterior and medial groups of thalamic nuclei. Stained axons were particularly concentrated in the ventrobasal complex, and in the stria medullaris, stria terminalis and inferior thalamic peduncle. The arrangement and density of labeled boutonlike dots varied markedly among nuclei, the highest densities occurring in the paraventricular and parataenial nuclei, and in the ventral subdivision of the lateral geniculate body. The mean staining intensity of the thalamic neuropil was lower than that of nearby structures, such as the hypothalamus and zona incerta. The present results on direct immunocytochemical detection of GABA are consistent with, and extend, data from immunocytochemical studies of the GABA-synthetizing enzyme, glutamic acid decarboxylase.

Slide-mounted 12-µm cryostat sections of rat brain were incubated with [3H]bilirubin, washed, dried, and apposed to emulsion-coated coverslips for autoradiography. The binding appeared to be nonsaturable. Within the gray matter, binding was concentrated over neuronal cell bodies (particularly evident over hippocampal pyramidal and granular cells, and over cerebellar Purkinje cells), suggesting that the preferential neuronal toxicity of bilirubin may be related to neuronal binding. Regional differences in bilirubin binding were not demonstrated.

TAURINE, AND glutamate are widely distributed amino acids in the mammalian brain, including the olfactory bulb where their functions have not been fully elucidated. This study investigated the precise cellular distribution of taurine, and glutamate in the nerve layer, and the glomeruli of the olfactory bulb, using semiquantitative immunocytochemistry at the electron microscopic level. The results show that both amino acids are present at higher concentrations in primary olfactory neurones than in the postsynaptic dendrites. Glutamate is also enriched in terminals vsaxons of the primary olfactory neurones. This suggests that glutamate acts as a neurotransmitter at the primary synapse in the olfactory system. An opposite concentration gradient is found for taurine, with a higher level of taurine-like immunoreactivity in axons than in terminals of primary olfactory neurones. Further studies are required before conclusions can be drawn about the function of taurine in these neurones.

Immunocytochemical methods for peptides and serotonin have greatly advanced the study of neurones in which these substances are likely to be transmitters1,2. Such direct techniques have not so far been available for the amino acid transmitter candidates. We report here the selective immunocytochemical visualization of the putative transmitters glutamate (Glu) and γ-aminobutyrate (GABA) by the use of antibodies raised against the amino acids coupled to bovine serum albumin (BSA) with glutaraldehyde (GA). The tissue localizations of Glu-like and GABA-like immunoreactivities (Glu-LI and GABA-LI) matched those of specific uptake sites for Glu and GABA, and, in the case of GABA-LI, also that of the specific marker enzyme glutamic acid decarboxylase (GAD). Thus, GABA-LI was located in what are believed to be GABAergic inhibitory neurones, whereas Glu-LI was concentrated in excitatory, possibly glutamatergic neurones. Preliminary electron microscopic observations suggest that the transmitter amino acids are significantly concentrated in synaptic vesicles.

PREVIOUS electrophysiological and pharmacological studies have suggested the involvement of both glycine and gamma-aminobutyric acid (GABA) as transmitters in the reflex pathway mediating recurrent inhibition of cat spinal motoneurones. By use of the postembedding immunogold technique and antibodies against glutaralde-hyde-conjugated amino acids it is shown that glycine- and GABA-immunoreactive nerve terminals occur in contact with cell bodies of alpha motoneurone size, as well as in the surrounding neuropil in the motor nuclei of the cat spinal cord. A substantial proportion of these terminals harbours both glycine- and GABA-LI. The enrichment of immunolabelling over synaptic vesicles in such terminals strongly suggests that both of these amino acids are used as transmitter substances.

The glutamate transporters GLT-1 and GLAST were studied by immunogold labeling on ultrathin sections of rat brain tissue embedded in acrylic resins at low temperature after freeze substitution. Both proteins were selective markers of astrocytic plasma membranes. GLT-1 was much higher in hippocampal astrocytes than in cerebellar astrocytes. Astroglial membrane GLAST densities ranked as follows: Bergmann > cerebellar granular layer ≈ hippocampus > cerebellar white matter. No astrocyte appeared unlabeled. Astrocytic membranes facing capillaries, pia, or stem dendrites were lower in glutamate transporters than those facing nerveterminals, axons, and spines. Parallel fiber boutons (glutamatergic) synapsing on interneuron dendritic shafts were surrounded by lower transporter densities than those synapsing on Purkinje cell spines. Our findings suggest the localizations of glutamate transporters are carefully regulated.

Human retinae from surgical specimens rapidly fixed in a glutaraldehyde/formaldehyde mixture were subjected to postembedding, immunogold immunocytochemistry of glutamate and glycine, and subsequently analysed in an electron microscope. The two amino acids were visualised in the same tissue sections by the use of two different gold particle sizes. All bipolar cell perikarya and terminals showed significant glutamate labelling with mean gold particle densities 3–4 times higher than those of the retinal, non-neural pigment epithelial and Müller cells. Bipolar cell terminals displayed significantly higher glutamate labelling density than the bipolar cell bodies, as would be expected of glutamatergic neurons. A subpopulation of the glutamate-immunolabelled bipolar cell bodies (18%) and terminals (32%) also exhibited strong glycine labelling (7–8 times that of pigment epithelial and Müller cells). These glutamate-glycine positive terminals established contacts with amacrine cell processes and ganglion cell dendrites and were localised almost exclusively at between 44% and 88% depth of the inner plexiform layer, indicating that they belong to the “ON” cone bipolar system. This subpopulation of terminals was endowed with significantly higher glycine labelling density than the glycine positive bipolar cell bodies. These results show that human bipolar cell terminals colocalise glutamate and glycine and provide the first direct demonstration of an enrichment of these two amino acids in the same presynaptic element.

Background/Aims:The inhibitory neurotransmitter γ-aminobutyrate (GABA) has been shown to coexist with insulin in pancreatic β-cells. We have presently investigated whether GABA also colocalizes with gastrin in G cells in rat antral mucosa. Methods:Three alternative approaches were used: (1) gastrin in situ hybridization and GABA immunocytochemistry on consecutive cryostat sections; (2) GABA immunocytochemistry and gastrin immunocytochemistry on adjacent semithin and ultrathin sections; and (3) double-immunogold labeling of GABA and gastrin in the same ultrathin section. Results:Colocalization of GABA and gastrin was observed with each of the three approaches. In the double-immunogold labeled cells, the G-cell granules displayed a high gold-particle density indicating gastrin and a low particle density indicating GABA, whereas the converse was true for the extragranular cytoplasmic matrix. The gold-particle ratios between these compartments were 11 (for gastrin) and 0.36 (for GABA), respectively. GABA labeling was also observed in two other antral endocrine cell types, classified by morphological criteria as somatostatin producing D cells and serotonin producing ECn cells. Conclusions:This is the first direct demonstration of GABA in gastrointestinal G cells. Our findings suggest that GABA may have a paracrine function in the stomach mucosa, analogous to its presumed role in the pancreatic islets.

GLUTAMIC ACID (glu) may be the transmitter of a large proportion of excitatory synapses in the brain1, but glu is also important in cell metabolism, and the apparent lack of biochemical ‘markers’ associated with the role of glu as a synaptic transmitter has until recently hampered the localisation of potential ‘glutamergic’ neurones. High affinity uptake of glu2,3is, however, highly specific2and seems to be selectively localised in the excitatory granular cell terminals in the cerebellum4, and in three systems of excitatory nerve endings in the hippocampal formation, as shown by autoradiography5and quantitative measurements6. In the conditions used in these studies, glial uptake of glu7,8was found not to be quantitatively important. In the hippocampal systems, iontophoretic studies9and release experiments10strongly suggest that glu, and/or aspartic acid (asp), may be the transmitter. It thus seems that high affinity uptake of glu may be useful as a marker for putative glutamergic and/or aspartergic nerve endings. The question of whether the uptake of [3H]glu, as measured in vitro, represents net accumulation or homoexchange11is not crucial in the present context. The neostriatum (nucleus caudatus–putamen) receives a large excitatory projection from the neocortex12,13. Iontophoretic studies suggest that this could use glu or asp as its transmitter1,14. We here demonstrate that high affinity uptake of glu is selectively reduced in the neostriatum after lesions in the neocortex.