No abstract

Morphine withdrawal is characterized by functional alterations at the level of the ventrotegmental area. We investigated the effects of chronic morphine administration and withdrawal on the morphological properties of immuno-labelled tyrosine hydroxylase-positive neurons of the rat ventrotegmental area with a confocal laser scanning microscope. Morphological evaluation revealed a reduction in the area and perimeter of tyrosine hydroxylase-positive somata in morphine-withdrawn rats. Conversely, the number of cells per field was found to have increased in the naloxone group. Collectively, the present results indicate that withdrawal from a chronic morphine treatment, and not chronic morphine per se, modifies cellular morphology of tyrosine hydroxylase-positive, presumably dopamine-containing, neurons of the rat VTA. This is consistent with the idea that withdrawal from morphine alters functioning of the mesolimbic dopamine system and provides a direct morphological correlate for the functional abnormalities typical of morphine withdrawal.

Morphine withdrawal produces a hypofunction of mesencephalic dopamine neurons that impinge upon medium spiny neurons (MSN) of the forebrain. After chronic treatment (from 20 to 140 mg/kg of morphine twice a day over 14 days at escalating doses) rats were withdrawn from chronic morphine spontaneously and pharmacologically. In these two distinct conditions we studied the effects of withdrawal on the morphology of MSN of the core and shell of the nucleus accumbens (Nacc). MSN were stained with the Golgi-Cox procedure and analysed by a confocal laser-scanning microscope (CLSM). Our analysis shows that, shell and core MSN differed significantly for perikarya size and spine density, and the various morphine treatments did not affect the perikarya morphometry. Both spontaneous and naloxone-induced withdrawal produced a similar reduction in spine density in MS shell neurons, as compared with MS core neurons. This effect is selectively localized at the level of second order dendritic trunks where afferents converge. By contrast, spine density counts of accumbens MSN from rats chronically treated with morphine, did not reveal any change. Collectively, the results of the present study are twofold: (i) spontaneous and pharmacologically precipitated withdrawal, but not chronic morphine per se, affects spine density of target structures of a reduced mesolimbic dopamine transmission, and (ii) the reduction of spine density in second order dendritic trunks is selectively segregated in the MSN of the shell of the Nacc. In conclusion, morphine withdrawal dramatically alters spine density, selectively in second order dendritic trunks of Nacc shell MSN, thereby further impoverishing the already abated dopamine (DA) transmission. This is in line with recent views suggesting the hypodopaminergic state as a cardinal feature of opioid dependence.

Visualization of neuronal elements is of fundamental importance in modern neuroscience. Golgi-Cox impregnation is a widely employed method that provides detailed information about morphological characteristics of neurons, but none regarding their neurochemical features. Immunocytochemical procedures, on the other hand, can provide a high degree of biochemical specificity but poorer morphological details, in particular if compared to Golgi-Cox impregnation. Hence, the combined use of these two approaches is highly desirable, especially for confocal microscopy that can exploit the advantages of both methods simultaneously. Here we show an innovative procedure of perfusion and fixation of brain tissue, that allows, by applying Golgi-Cox impregnation and immunofluorescence in the same histological section, to obtain high-quality histological material, with a very simple and inexpensive method. This procedure is based on three simple fixation steps: (1) a paraformaldehyde perfusion followed by a standard post-fixation to stabilize the subsequent immunofluorescence reaction; (2) the classical Golgi-Cox impregnation and (3) an immunofluorescence reaction in previously impregnated material. This combination allows simultaneous visualization of (a) the structural details (Golgi-Cox impregnated neurons), (b) the antigens’ characterization, (c) the anatomical interactions between discrete neuronal elements and (d) the 3D reconstruction and modeling. The method is easy to perform and can be reproducibly applied by small laboratories and expanded through the use of different antibodies. Overall, the method presented in this study offers an innovative and powerful approach to study the nervous system, especially by using confocal microscopy.

This symposium focused on functional alterations in the mesolimbic dopamine system during the abstinence phase after chronic alcohol intake. Mark Brodie first described his recordings from midbrain slices prepared after chronic alcohol treatment in vivo by daily injection in C57BL/6J mice. No changes were found in the baseline firing frequency of dopaminergic neurones in the VTA (ventral tegmental area), but the excitation produced in these neurones by an acute ethanol challenge was significantly increased in neurons from ethanol-treated mice compared with those from the saline-treated controls. There was also a significant decrease in the inhibitory response to GABA by the dopamine neurones following the chronic ethanol treatment. These data suggest that the timing pattern and mode of ethanol administration may determine the types of changes observed in dopaminergic reward area neurons. Annalisa Muntoni lectured on the relationship between electrophysiological and biochemical in vivo evidence supporting a reduction in tonic activity of dopamine neurons projecting to the nucleus accumbens at various times after suspension of chronic ethanol treatment and morphological changes affecting dopamine neurons in rat VTA. Hilary J. Little then described changes in dopaminergic neurone function in the VTA during the abstinence phase. Decreases in baseline firing were seen at 6 days after withdrawal of mice from chronic ethanol treatment but were not apparent after 2 months abstinence. Increases in the affinity of D1 receptors in the striatum, but not in the cerebral cortex, were seen however up to 2 months after withdrawal. Scott Steffensen then described his studies recording in vivo from GABA containing neurones in the VTA in freely moving rats. Chronic ethanol administration enhanced the baseline activity of these neurones and resulted in tolerance to the inhibition by ethanol of these neurones. His results demonstrated selective adaptive circuit responses within the VTA or in extrategmental structures that regulate VTA-GABA neurone activity.