Three-dimensional computer reconstructions from serial sections of cell nuclei

The analysis of ultrathin serial sections as 3-dimensional (3D) information requires interpretation and display of a large amount of data. We suggest a simple way to solve this problem; it permits presentation of a series of sections as a 3D color image of good quality. It involves a picture system with specialized hardware and software written for this purpose. 3D images of cellular organelles have been drawn either by manually defining the contour of the objects or by thresholding of the volumes in the structures. These 2 methods allow rapid drawing of the image on the screen. It is possible to determine the position, shape and size of 3D structures. This interactive system allows the user to choose between several options: colors, removal of parts of the object, and cutout.     

Ultrastructural studies on the hypothalamic neurosecretory neurones of the rat

Summary The ultrastructural features of the paraventricular neurones of the non-treated rat are presented comparing them with those of the supraoptic neurones. No striking differences are seen between the general electron microscopic characteristics of the paraventricular and supraoptic neurones.The importance of adequate fixation to obtain good preservation of the neurones is emphasized, since inadequate fixation can cause e.g. artefactual appearance of dark neurones. The previously presented classification of the neurosecretory neurones into two categories (e.g. light and dark neurones) on the basis of the number of ribosomes is not considered justifiable, since their number can vary to a very great extent even within a single cell.The synthesis of neurosecretory products in the paraventricular neurones obviously follows the general mode of the synthesis of secretory proteins: ribosomes—RER—Golgi complex—secretory vesicles.On the basis of the localization of heavy metal deposits after osmium impregnation and demonstration of acid phosphatase the Golgi complex of the paraventricular neurones is found to be polarized. The direction of the polarity is discussed.The substructures of the dense cores of the neurosecretory granules and of the contents of the lysosomal dense bodies are nearly identical. Therefore it is considered impossible to determine positively the nature of the dark condensed material within the Golgi complex. The characteristics of the immature neurosecretory granules and the possibility of releasing neurosecretory products into the cytoplasm already within the perikarya are speculated.This study was supported by a grant from the Emil Aaltonen Foundation, Tampere. I express my best thanks to Docent Antti U. Arstila, Head of the Laboratory of Electron Microscopy, and Professor Urpo K. Rinne, Head of the Department of Neurology, for the guidance of this work.     

Involvement of the K+‐Cl− co‐transporter KCC2 in the sensitization to morphine‐induced hyperlocomotion under chronic treatment with zolpidem in the mesolimbic system

Benzodiazepines are commonly used as sedatives, sleeping aids, and anti‐anxiety drugs. However, chronic treatment with benzodiazepines is known to induce dependence, which is considered related to neuroplastic changes in the mesolimbic system. This study investigated the involvement of K⁺‐Cl^? co‐transporter 2 (KCC2) in the sensitization to morphine‐induced hyperlocomotion after chronic treatment with zolpidem [a selective agonist of γ‐aminobutyric acid A‐type receptor (GABA_AR) α1 subunit]. In this study, chronic treatment with zolpidem enhanced morphine‐induced hyperlocomotion, which is accompanied by the up‐regulation of KCC2 in the limbic forebrain. We also found that chronic treatment with zolpidem induced the down‐regulation of protein phosphatase‐1 (PP‐1) as well as the up‐regulation of phosphorylated protein kinase C γ (pPKCγ). Furthermore, PP‐1 directly associated with KCC2 and pPKCγ, whereas pPKCγ did not associate with KCC2. On the other hand, pre‐treatment with furosemide (a KCC2 inhibitor) suppressed the enhancing effects of zolpidem on morphine‐induced hyperlocomotion. These results suggest that the mesolimbic dopaminergic system could be amenable to neuroplastic change through a pPKCγ‐PP‐1‐KCC2 pathway by chronic treatment with zolpidem.     

The GABAergic network in the suprachiasmatic nucleus as a key regulator of the biological clock: does it change during senescence?

GABA is the main neurotransmitter of the hypothalamic suprachiasmatic nucleus (SCN) and plays a key role in the function of this master circadian pacemaker. Despite the evidence that disturbances of biological rhythms are common during aging, little is known about the GABAergic network in the SCN of the aging brain. We here provide a brief overview of the GABAergic structures and the role of GABA in the SCN. We also review some age‐related changes of the GABAergic system occurring in the brain outside the SCN. Finally, we present preliminary data on the GABAergic system within the SCN comparing young and aging mice. In particular, our study on age‐related changes in the SCN focused on the daily expression of the α3 subunit of the GABA_A receptor and on the density of GABAergic axon terminals. Interestingly, our preliminary findings point to alterations of the GABAergic network in the biological clock during senescence.     

Suprachiasmatic Nuclei May Regulate the Rhythm of Leptin Hormone Release in Syrian Hamsters (Mesocricetus Auratus)

The suprachiasmatic nuclei (SCN) generate the circadian rhythm of many hormones. The hormone leptin is a metabolic signal that informs the brain about fat and energy stores of the body. We investigated whether the rhythm of leptin hormone release in Syrian hamsters is directly controlled by the SCN. Three experiments were performed: in the first, hamsters were SCN‐lesioned; in the second, hamsters were exposed to different feeding regimes; and in the third, hamsters were adrenalectomized and implanted with cortisol capsules to maintain constant glucocorticoid release. Blood samples were collected before and after the experiments at different clock times and examined for leptin levels by enzyme‐linked immunosorbant assay (ELISA). Different feeding regimes and constant glucocorticoid release did not alter the rhythm of leptin release; whereas, SCN lesions abolished the rhythm. The results of the present study suggest the rhythm in leptin release in Syrian hamsters may be controlled by the SCN.     

Nicotinic acetylcholine receptors containing α6 subunits contribute to alcohol reward‐related behaviours

Evidence is emerging that neuronal nicotinic acetylcholine receptors (nAChRs) in the mesolimbic dopamine (DA) system are involved in mediating the reinforcing effects of alcohol. Midbrain DA neurons express high levels of α6 subunit‐containing nAChRs that modulate DA transmission, implicating their involvement in reward‐related behaviours. This study assessed the role of α6‐containing nAChRs in modulating alcohol reward using transgenic mice expressing mutant, hypersensitive α6 nAChR subunits (α6L9′S mice). α6L9′S mice and littermate controls were tested in three well‐established models of alcohol reward: 24‐h two‐bottle choice drinking, drinking in the dark (DID), and conditioned place preference (CPP). Confocal microscopy and patch‐clamp electrophysiology were used to show the localization and function of hypersensitive α6 subunit‐containing nAChRs. Results indicate that female α6L9′S mice showed significantly higher alcohol intake at low concentrations of alcohol (3% and 6%) in the two‐bottle choice procedure. Both male and female α6L9′S mice drank significantly more in the DID procedure and displayed an alcohol‐induced place preference using a low dose of alcohol (0.5 g/kg) that was ineffective in littermate controls. Confocal microscopy showed that α6 subunit‐containing nAChRs are selectively expressed on ventral tegmental area (VTA) DAergic, but not GABAergic neurons. Patch‐clamp electrophysiology showed that VTA DA neurons of α6L9′S mice are hypersensitive to ACh. Collectively, these results suggest that α6L9′S mice are more sensitive to the rewarding effects of alcohol, and suggest that VTA α6 subunit‐containing nAChRs modulate alcohol reward. Thus, α6 subunit‐containing nAChRs may be a promising therapeutic target for treatment of alcohol use disorders .     

Drug-primed reinstatement of cocaine seeking in mice: increased excitability of medium-sized spiny neurons in the nucleus accumbens

To examine the mechanisms of drug relapse, we first established a model for cocaine IVSA (intravenous self-administration) in mice, and subsequently examined electrophysiological alterations of MSNs (medium-sized spiny neurons) in the NAc (nucleus accumbens) before and after acute application of cocaine in slices. Three groups were included: master mice trained by AL (active lever) pressings followed by IV (intravenous) cocaine delivery, yoked mice that received passive IV cocaine administration initiated by paired master mice, and saline controls. MSNs recorded in the NAc shell in master mice exhibited higher membrane input resistances but lower frequencies and smaller amplitudes of sEPSCs (spontaneous excitatory postsynaptic currents) compared with neurons recorded from saline control mice, whereas cells in the NAc core had higher sEPSCs frequencies and larger amplitudes. Furthermore, sEPSCs in MSNs of the shell compartment displayed longer decay times, suggesting that both pre- and postsynaptic mechanisms were involved. After acute re-exposure to a low-dose of cocaine in vitro, an AP (action potential)-dependent, persistent increase in sEPSC frequency was observed in both NAc shell and core MSNs from master, but not yoked or saline control mice. Furthermore, re-exposure to cocaine induced membrane hyperpolarization, but concomitantly increased excitability of MSNs from master mice, as evidenced by increased membrane input resistance, decreased depolarizing current to generate APs, and a more negative Thr (threshold) for firing. These data demonstrate functional differences in NAc MSNs after chronic contingent versus non-contingent IV cocaine administration in mice, as well as synaptic adaptations of MSNs before and after acute re-exposure to cocaine. Reversing these functional alterations in NAc could represent a rational target for the treatment of some reward-related behaviors, including drug addiction.