Revaz Solomonia

Glial and neuronal opioid receptors: apparent positive cooperativity observed in intact cultured cells.

Opioid receptors were characterized in glial and neuronal homogeneous cultures of embryonic chick forebrain, using [3H]naloxone as a labelled ligand. Binding experiments were performed on intact cells. The specific binding of [3H]naloxone reached equilibrium after 1 min. The apparent dissociation constants were estimated as 0.51 nM for glial and 0.63 nM for neuronal cells. Equilibrium measurements indicated the apparent positive cooperativity of the binding, resulting in Hill coefficients of 2.61 for glial and 2.04 for neuronal cells. Competition of unlabelled naloxone for specific binding sites resulted in maximum-shape curves in glial cells if measured at low receptor occupancy. This supports the positive cooperativity of ligand binding. Opioid agonists, ethylketocyclazocine (EKC), morphine and [D-Ala2,L-Leu5]enkephalin (DALA), provoked biphasic competition curves in both cell types with a characteristic maximum at low competitor concentrations. The possible physiological role of glial opioid receptors in neuron-glia communication and the significance of cooperativity is discussed.

Alterations of GABA(A) and glutamate receptor subunits and heat shock protein in rat hippocampus following traumatic brain injury and in posttraumatic epilepsy.

Traumatic brain injury (TBI) can result in the development of posttraumatic epilepsy (PTE). Recently, we reported differential alterations in tonic and phasic GABA(A) receptor (GABA(A)R) currents in hippocampal dentate granule cells 90 days after controlled cortical impact (CCI) (Mtchedlishvili et al., 2010). In the present study, we investigated long-term changes in the protein expression of GABA(A)R α1, α4, γ2, and δ subunits, NMDA (NR2B) and AMPA (GluR1) receptor subunits, and heat shock proteins (HSP70 and HSP90) in the hippocampus of Sprague-Dawley rats evaluated by Western blotting in controls, CCI-injured animals without PTE (CCI group), and CCI-injured animals with PTE (PTE group). No differences were found among all three groups for α1 and α4 subunits. Significant reduction of γ2 protein was observed in the PTE group compared to control. CCI caused a 194% and 127% increase of δ protein in the CCI group compared to control (p<0.0001), and PTE (p<0.0001) groups, respectively. NR2B protein was increased in CCI and PTE groups compared to control (p=0.0001, and p=0.011, respectively). GluR1 protein was significantly decreased in CCI and PTE groups compared to control (p=0.003, and p=0.001, respectively), and in the PTE group compared to the CCI group (p=0.036). HSP70 was increased in CCI and PTE groups compared to control (p=0.014, and p=0.005, respectively); no changes were found in HSP90 expression. These results provide for the first time evidence of long-term alterations of GABA(A) and glutamate receptor subunits and a HSP following CCI.

Neural cell adhesion molecules, learning, and memory in the domestic chick

The intermediate and medial hyperstriatum ventrale (IMHV) of the chick forebrain is a site of recognition memory for the learning process of imprinting. The results reported here demonstrate that neural cell adhesion molecules (NCAMs) play a time-dependent role in this recognition memory. Dark-reared chicks were trained, tested, and assigned a preference score as a measure of learning. Chicks with high preference scores were designated good learners and those with lower preference scores, poor learners. Controls were untrained. Tissue was removed, 9.5 hr or 24 hr after training, from the left and right IMHV, hyperstriatum accessorium, and posterior neostriatum. Three major NCAM isoforms (180, 140, and 120 kDa) were assayed. At 24 hr only, there was in left IMHV significantly more NCAM (for each isoform) in good learners than in the other 2 groups, and also a significant correlation between the amounts of NCAM and preference scores for all isoforms; the amount predicted by each regression line at preference score 50 (no learning) did not differ significantly from the mean value for untrained controls. There were no learning-related effects in either the hyperstriatum accessorium or the posterior neostriatum.

Differential distribution of protein kinase C (PKCαβ and PKCγ) isoenzyme immunoreactivity in the chick brain

Protein kinase C (PKC) is involved in neural plasticity. The phosphorylation of the myristoylated alanine-rich protein kinase C substrate (MARCKS) in the left intermediate and medial hyperstriatum ventrale (IMHV) of the chick brain has been shown previously to correlate significantly with the strength of learning in filial imprinting. The distribution of PKC alpha, beta I, beta II and PKC gamma in the brain of 1-day-old dark-reared chicks was determined immunocytochemically, using the monoclonal antibodies MC5 and 36G9, raised against purified PKC alpha beta and PKC gamma, respectively. PKC gamma-stained cells were distributed widely in the telencephalon, including all hyperstriatal structures (including the IMHV), the hippocampus, neostriatum, ectostriatum and archistriatum. There were fewer stained cells in the septum and the least cellular staining was in the paleostriatum primitivum. Fluorescent double-labelling with neuron-specific enolase (NSE) and with the glial calcium-binding protein S100 suggested that PKC gamma immunoreactivity was present in neurones but not in glia. The distribution of PKC alpha beta-stained cells was more limited, with staining in the archistriatum, hippocampus and septum but not in the hyperstriatum. However, there was PKC alpha beta-staining of some fibres in the IMHV (but little elsewhere in the hyperstriatum ventrale), in the neostriatum, paleostriatal complex and the lobus parolfactorius. Double-labelling with NSE and S100 revealed PKC alpha beta/S100-positive glial cells present in the paleostriatal region only. There was some PKC alpha beta-staining of putative neurones in the hippocampus, septum and archistriatum. The differential distribution of PKC isoenzymes suggests that in the IMHV some axonal inputs contain PKC alpha beta whereas some postsynaptic cells contain the gamma form of PKC.

Analysis of differential gene expression supports a role for amyloid precursor protein and a protein kinase C substrate (MARCKS) in long-term memory

Previous work has identified the intermediate and medial part of the hyperstriatum ventrale (IMHV) as a region of the chick brain storing information acquired through the learning process of imprinting. We have examined in this brain region changes in expression of candidate genes involved in memory. Chicks were exposed to a rotating red box and the strength of their preference for it, a measure of learning, determined. Brain samples were removed ≈24 h after training. Candidate genes whose expressions were different in IMHV samples derived from strongly imprinted chicks relative to those from chicks showing little or no learning were identified using subtractive hybridization. The translation products of two candidate genes were investigated further in samples from the left and right IMHV and from two other brain regions not previously implicated in imprinting, the left and right posterior neostriatum. One of the proteins was the amyloid precursor protein (APP), the other was myristoylated alanine rich C kinase substrate (MARCKS). In the left IMHV the levels of the two proteins increased with the strength of learning. The effects in the right IMHV were not significantly different from those in the left. There were no effects of learning in the posterior neostriatum. This is the first study to relate changes in the amounts of MARCKS and APP proteins to the strength of learning in a brain region known to be a memory store and demonstrates that the systematic identification of protein molecules involved in memory formation is possible.

Ca2+/calmodulin protein kinase II and memory: learning-related changes in a localized region of the domestic chick brain

The role of calcium/calmodulin‐dependent protein kinase II (CaMKII) in the recognition memory of visual imprinting was investigated. Domestic chicks were exposed to a training stimulus and learning strength measured. Trained chicks, together with untrained chicks, were killed either 1 h or 24 h after training. The intermediate and medial hyperstriatum ventrale/mesopallium (IMHV/IMM), a forebrain memory storage site, was removed together with a control brain region, the posterior pole of the neostriatum/nidopallium (PPN). Amounts of membrane total αCaMKII (tCaMKII) and Thr286‐autophosphorylated αCaMKII (apCAMKII) were measured. For the IMHV/IMM 1 h group, apCaMKII amount and apCAMKII/tCaMKII increased as chicks learned. The magnitude of the molecular changes were positively correlated with learning strength. No learning‐related effects were observed in PPN, or in either region at 24 h. These results suggest that CaMKII is involved in the formation of memory but not in its maintenance.

Myo-inositol treatment prevents biochemical changes triggered by kainate-induced status epilepticus

Identification of the compounds preventing the biochemical changes underlying the epileptogenesis process is of great importance. We have previously shown that myo-inositol (MI) administration reduces kainic acid (KA) induced seizure scores. MI treatment effects on biochemical changes triggered by KA induced status epilepticus (SE) were investigated in the present study. After SE one group of rats was treated with saline, whereas the second group with MI. Control groups received either saline or MI administration. Changes in the amounts of following proteins were studied in the hippocampus and neocortex of rats: GLUR1 subunit of glutamate receptors, calcium/calmodulin-dependent protein kinase II (CaMKII), and heat shock protein 90. No changes were found 28-30h after experiments. However on 28th day of experiment the amounts of GLUR1 and CaMKII were strongly reduced in the hippocampus of KA treated animals but MI significantly halted this reduction. Obtained results indicate anti-epileptogenic features of MI on biochemical level.

Anticonvulsant activities of myo-inositol and scyllo-inositol on pentylenetetrazol induced seizures

Myo-inositol (MI) and its isomers are used for the treatment of various neuropathological conditions. The purpose of the present research was to study anticonvulsant properties of MI and scyllo-inositol (SCI) on pentylenetetrazol (PTZ) induced seizures in rats. Half an hour after treatment with MI (30 mg/kg) or SCI (5 mg/kg) seizures were induced in Wistar rats with PTZ (60 mg/kg). Control animals were treated either by normal saline or mannitol (control polyol of the same molecular weight, 30 mg/kg), given at the same time interval before PTZ injection, as MI/SCI groups. The anticonvulsant effects of MI/SCI treatment were assessed by the latent period (the time from PTZ-injection to the onset of first seizures), and the duration and severity (score) of seizures. The mortality rate was also assessed. Both MI and SCI treatment significantly reduced the seizure score, seizure duration and increased the latent period. These data suggest for strong potential of MI and SCI as the agents of antiepileptic therapy.

Expression of the GABA(A) receptor gamma 4-subunit gene: anatomical distribution of the corresponding mRNA in the domestic chick forebrain and the effect of imprinting training.

The learning process of imprinting involves morphological, electrophysiological and biochemical changes in a region of the chick (Gallus gallus domesticus) forebrain known as the intermediate and medial part of the hyperstriatum ventrale (IMHV). The alterations include increases in the mean length of postsynaptic density profiles of axospinous synapses and the number of N‐methyl‐d‐aspartate (NMDA) receptor binding sites, and changes in spontaneous and evoked electrical activity. Recent immunocytochemical and behavioural studies have suggested that inhibitory GABAergic neurotransmission plays a role in learning. In this context, it has previously been reported that a novel avian γ‐aminobutyric acid (GABA) type A (GABAA) receptor gene, encoding the γ4 subunit, is highly expressed in the hyperstriatum ventrale. In this study, we have used in situ hybridization to map, in detail, the expression of the γ4‐subunit gene in the chick brain, and to assess the effect of imprinting training on the level of the corresponding transcript. Our results reveal that the γ4‐subunit mRNA has a restricted distribution, and demonstrate a highly significant, time‐dependent effect of training on its steady‐state level. At 10 h but not at 5 h after training there is a decrease (25–32%) in the amount of this transcript in parts of the medial hyperstriatum ventrale, including the IMHV. A decrease (28–39%) is also seen in certain visual and auditory pathway areas but no effect was observed in other forebrain regions such as the hyperstriatum intercalatus superior (HIS). These results suggest that imprinting training leads to a time‐dependent down‐regulation of GABAergic transmission, and raise the possibility that this down‐regulation plays a role in learning.

Molecular mechanisms of memory in imprinting

Highlights • We discuss learning-related biochemical changes in a chick brain memory system.• These changes reflect neuronal responsiveness to the imprinting stimulus.• Early changes occur in c-fos, synaptic protein phosphorylation and transmitter pool.• Intermediate changes involve transmitter pool and glutamate NMDA receptors.• Late changes (with maximum neuronal responsiveness) involve protein synthesis.