William S. Messer

A simple spatial alternation task for assessing memory function in zebrafish.

A series of studies was initiated to examine learning and memory function in the zebrafish (Danio rerio) by using a simple spatial alternation paradigm for a food reward. Fish were fed on alternating sides of a divided fish tank, with a red card displayed on one side serving as a visual means of orientation. Although responses were recorded at cue (light tap on the tank), 5 s after cue (as food was delivered), and 5 s after food delivery, the learning test was choice of a correct side of the tank to receive food. Therefore, an accurate level of an animals achievement of the spatial task was represented by responses at food delivery. Data collected from 11 separate experiments indicated that zebrafish learned to alternate for a food reward. Further, statistical analysis showed that the zebrafish learned the task in the first half of the experiment as exhibited by a calculated t1/2 of 13.9 trials. Zebrafish could recall the task after a short period of 10 days with no testing. The alternating behavior was extinguished by withholding the food reward. Thus, the spatial alternation task can be learned easily by zebrafish, and may be useful in addressing learning and memory functions in vertebrate animals using zebrafish as a model organism.

Evidence for a preferential involvement of M1 muscarinic receptors in representational memory

The effects of intrahippocampal injections to the M1-selective antagonist pirenzepine and the M2-selective antagonist AF-DX 116 were examined on performance of a representational memory task in rats. Although both antagonists impaired performance, pirenzepine was more potent than AF-DX 116. Pirenzepine (70.8 +/- 2.8% correct) produced a greater deficit than AF-DX 116 (83.3 +/- 0.0%) at 70 micrograms, and the deficit at 10 micrograms (83.3 +/- 2.8%) was equal to that produced by 70 micrograms of AF-DX 116. The data provide additional support for the cholinergic hypothesis of memory and new information regarding the subtypes of muscarinic receptors likely to be involved in representational memory. Based on the greater susceptibility of representational memory to the effects of pirenzepine, it is suggested that M1 receptors in the hippocampus play a greater role in memory function than M2 receptors.

Biochemical and behavioral responses of pilocarpine at muscarinic receptor subtypes in the CNS. Comparison with receptor binding and low-energy conformations

Pilocarpine was tested biochemically in vitro for its ability to stimulate phosphoinositide (PI) turnover in the hippocampus (M1/M3 responses) where it displayed 35% of the maximal carbachol response with an EC50 value of 18 microM, and low-Km GTPase in the cortex (M2 response), where it had 50% of the maximal carbachol response with an EC50 value of 4.5 microM. Behaviorally, pilocarpine was able to restore deficits in a representational memory task (sensitive to M1 antagonists) produced by intrahippocampal injections of AF64A. Twenty-three low-energy conformations of protonated pilocarpine were generated using the program MacroModel. The data indicate that pilocarpine is a partial agonist at both M1 and M2 muscarinic receptors in the CNS. Behaviorally, with respect to the memory task, M1 effects of pilocarpine apparently predominate. It also is conceivable that different conformations of pilocarpine are active as agonists at different muscarinic receptor subtypes.

Differential coupling between muscarinic receptors and G-proteins in regions of the rat brain

The coupling of muscarinic receptors to G-proteins in various regions of the rat brain was assessed by measuring carbachol-stimulated, low-Km GTPase. The inhibition of carbachol-stimulated GTPase by the M1-selective antagonist pirenzepine was compared to the affinity of pirenzepine for various nuclei within the regions as measured autoradiographically. The rank order of potency of carbachol for stimulating GTPase in various brain regions was similar to that for binding to receptors in those areas. The maximal specific activity (efficacy) of carbachol-stimulated GTPase varied independently of the distribution of total receptors or receptor subtypes. The overall potency of pirenzepine for inhibiting carbachol-stimulated GTPase was not correlated with the overall affinity of pirenzepine for muscarinic receptors in the regions. Comparing results in various brain regions, the data suggest that there are differences in the efficiency of coupling between muscarinic receptors and G-proteins. For example, the pons-medulla appeared to have a small population of pirenzepine-sensitive (M1 or M4) receptors that were coupled very efficiently to G-proteins, whereas in the hippocampus all muscarinic receptors, most of which are pirenzepine-sensitive, appeared to be weakly coupled to G-proteins. It is suggested that variable interactions between receptors and G-proteins may be an important factor in the overall coupling between receptor occupancy and cellular responses to acetylcholine as well as other hormones and transmitters.

Involvement of the septohippocampal cholinergic system in representational memory

To develop an animal model for testing muscarinic agonists, we examined the effects of cholinergic lesions with the ethylcholine aziridinium ion (AF64A) on two types of memory tasks. The tasks provided a distinction between representational and dispositional memory that could be measured in a single paradigm. Young, male Long-Evans rats were trained in a modified T-maze to learn both a discrimination task and a paired-run alternation task. Once animals learned the tasks, they were administered either saline or AF64A (5 nmol into each hippocampus) via stereotaxic technique. One week following surgery, saline-treated animals exhibited comparable performances (P greater than 0.2) on both the discrimination task (90.0 +/- 2.6% correct) and the alternation task (79.5 +/- 5.7%). In contrast, animals treated with AF64A showed a significant impairment of performance (P less than 0.005) on the alternation task (56.1 +/- 1.7%) as compared to the discrimination task (81.6 +/- 5.0%). Performance of the alternation task was significantly lower for AF64A-treated animals than for controls (P less than 0.02). AF64A-treated animals subsequently injected with pilocarpine (1.0 mg/kg, i.p.) showed moderate improvements in performance on the alternation task, while performance on the discrimination task remained unaffected. Immunocytochemical studies of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) immunoreactivity indicated a loss of ChAT-positive cells in the septal region in AF64A-injected animals while TH-positive cells in the ventral tegmental area were unaffected by the treatment. The data suggest that AF64A can be used to produce selective lesions of the septohippocampal cholinergic system, which plays a greater role in representational memory than in dispositional memory.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiographic analyses of agonist binding to muscarinic receptor subtypes

The binding of four muscarinic receptor agonists to regions of rat brain was examined through quantitative autoradiographic techniques. Oxotremorine, arecoline, pilocarpine and bethanechol were chosen based on their different potencies and efficacies in muscarinic second messenger systems. Overall, the order of potency for inhibition of [3H]-l-quinuclidinyl benzilate ([3H]-l-QNB) binding to rat brain slices was oxotremorine greater than pilocarpine = arecoline much greater than bethanechol. Regional assays of agonist potency indicated that all agonists were more selective for brainstem and thalamic regions than for hippocampal and cortical regions. The high selectivity of agonists for areas such as the paraventricular thalamus and the superior colliculus, which also display low affinity for pirenzepine, suggests that muscarinic agonists bind with higher affinity to M2 receptors. Of the four agonists examined, pilocarpine displayed the lowest selectivity for M2 receptors in that IC50 values for pilocarpine were only 3-fold higher in the hippocampal and striatal regions (e.g. CA3: 40.6 +/- 9.4 microM) than in thalamic and brainstem regions (e.g. paraventricular thalamus: 14.9 +/- 6.2 microM). Oxotremorine was 8-fold more potent in the brainstem and thalamus, while arecoline and bethanechol were, respectively, 19- and 100-fold more selective for brainstem and thalamic receptors. Scatchard analyses revealed heterogeneous binding profiles for some agonists within single brain regions, suggesting that multiple agonist sites exist even within regions of predominantly M1 or M2 receptors. For example, arecoline displayed curved Scatchard plots within the external layers of the cerebral cortex, layer CA1 of the hippocampus (predominantly M1 subtype), and the paraventricular thalamus (predominantly M2 subtype). The ability of agonists to recognize multiple sites within a single region may reflect the ability to recognize receptors coupled or uncoupled to second messenger systems through G-proteins.

Intrahippocampal injections of gallamine impair learning of a memory task.

Recent evidence has suggested that gallamine may act as an antagonist on presynaptic M2 muscarinic receptors which regulate acetylcholine release. The possibility that gallamine may potentiate learning by enhancing the release of acetylcholine was examined using a behavioral paradigm known to depend on cholinergic activity. Rats were cannulated bilaterally for intrahippocampal (i.h.) injections and adapted to a T-maze before the series of injections was initiated. The effects of bilateral i.h. injections of saline or gallamine (10 micrograms total) were examined on the learning of a win-stay, paired-run task in a T-maze. During the first few sessions, both saline- and gallamine-treated animals achieved below 50% correct responses. Both groups of animals gradually improved performance on the task although, contrary to expectations, gallamine-treated animals did not learn as rapidly as saline-injected animals. To verify the deleterious effects of gallamine on learning, the experiment was repeated using a second group of rats. Similar results were observed; gallamine-treated animals learned to perform the task at a slower rate than the saline-injected controls. The results suggest that treatment strategies with cholinomimetics may impair, rather than enhance performance after long-term administrations.

Identification of four brain areas each enriched in a unique muscarinic receptor subtype

The affinities of muscarinic agonists and antagonists were determined by autoradiography and image analysis in selected areas of the rat brain. IC50 values and Hill coefficients for the inhibition of the binding of 0.2 nM [3H]-QNB to dentate gyrus, superior colliculus, rhomboid thalamus and substantia nigra were measured in coronal sections. Pirenzepine displayed a high affinity for receptors in the dentate gyrus and AF-DX 116, the superior colliculus. Both pirenzepine and AF-DX 116 had high affinities for the substantia nigra and low affinities for the rhomboid thalamus. Gallamine displayed a 50-fold preference for superior colliculus over dentate gyrus receptors. Amitriptyline was less selective, showing a modest preference for substantia nigra receptors and 4-DAMP was essentially nonselective. Carbachol was the most selective agonist with a 4000-fold preference for superior colliculus over dentate gyrus receptors. Other agonists except RS 86 were also selective for superior colliculus receptors in the order carbachol much greater than arecoline greater than bethanechol greater than McN A343 = oxotremorine = pilocarpine.

Multiple Protein Kinases Determine the Phosphorylated State of the Small Heat Shock Protein, HSP27, in SH-SY5Y Neuroblastoma Cells

In SH-SY5Y human neuroblastoma cells, the cholinergic agonist, carbachol, stimulates phosphorylation of the small heat shock protein 27 (HSP27). Carbachol increases phosphorylation of both Ser-82 and Ser-78 while the phorbol ester, phorbol-12, 13-dibutyrate (PDB) affects only Ser-82. Muscarinic receptor activation by carbachol was confirmed by sensitivity of Ser-82 phosphorylation to hyoscyamine with no effect of nicotine or bradykinin. This response to carbachol is partially reduced by inhibition of protein kinase C (PKC) with GF 109203X and p38 mitogen-activated protein kinase (MAPK) with SB 203580. In contrast, phosphorylation produced by PDB is completely reversed by GF 109203X or CID 755673, an inhibitor of PKD. Inhibition of phosphatidylinositol 3-kinase or Akt with LY 294002 or Akti-1/2 stimulates HSP27 phosphorylation while rapamycin, which inhibits mTORC1, does not. The stimulatory effect of Akti-1/2 is reversed by SB 203580 and correlates with increased p38 MAPK phosphorylation. SH-SY5Y cells differentiated with a low concentration of PDB and basic fibroblast growth factor to a more neuronal phenotype retain carbachol-, PDB- and Akti-1/2-responsive HSP27 phosphorylation. Immunofluorescence microscopy confirms increased HSP27 phosphorylation in response to carbachol or PDB. At cell margins, PDB causes f-actin to reorganize forming lamellipodial structures from which phospho-HSP27 is segregated. The resultant phenotypic change in cell morphology is dependent upon PKC, but not PKD, activity. The major conclusion from this study is that the phosphorylated state of HSP27 in SH-SY5Y cells results from integrated signaling involving PKC, p38 MAPK and Akt.

Steric and electronic requirements for muscarinic receptor-stimulated phosphoinositide turnover in the CNS in a series of arecoline bioisosteres

A series of arecoline derivatives was utilized to assess steric and electronic effects important for activating muscarinic receptors in the CNS. Arecoline derivatives in which the methyl ester moiety was replaced by hexyloxy-1,2,5-oxadiazole (2b), hexyloxythiophene (3b) or hexyloxypyrazine (4b) were compared with the hexyloxy-1,2,5-thiadiazole compound (1b) (Hexyloxy-TZTP), known from previous work to be active as an M1/M3 partial agonist. MNDO calculations showed that the N-S bonds of the alkoxythiadiazole ring were highly polarized with the ability to form H-bonds to the Ns. On the other hand, the smaller oxadiazole had lower polarities in the N-O bonds and reduced ability to form H-bonds, the thiophene was of comparable size to the thiadiazole and had large C-S bond polarities without the H-bond capability and the pyrazine had limited ability to form H-bonds. The compounds were compared with respect to their abilities to stimulate phosphoinositide (Pl) turnover in the hippocampus of the rat brain. 1b was more active than 2b-4b for stimulating the Pl turnover response. The data suggest that the ability to form H-bonds is an important factor for the ability of 1 to stimulate M1 muscarinic receptors in the CNS.