Susan E. Hamilton

Selective cognitive dysfunction in acetylcholine M1 muscarinic receptor mutant mice

Blockade of cholinergic neurotransmission by muscarinic receptor antagonists produces profound deficits in attention and memory. However, the antagonists used in previous studies bind to more than one of the five muscarinic receptor subtypes. Here we examined memory in mice with a null mutation of the gene coding the M1 receptor, the most densely distributed muscarinic receptor in the hippocampus and forebrain. In contrast with previous studies using nonselective pharmacological antagonists, the M1 receptor deletion produced a selective phenotype that included both enhancements and deficits in memory. Long-term potentiation (LTP) in response to theta burst stimulation in the hippocampus was also reduced in mutant mice. M1 null mutant mice showed normal or enhanced memory for tasks that involved matching-to-sample problems, but they were severely impaired in non-matching-to-sample working memory as well as consolidation. Our results suggest that the M1 receptor is specifically involved in memory processes for which the cortex and hippocampus interact.

Cholinergic Suppression of KCNQ Channel Currents Enhances Excitability of Striatal Medium Spiny Neurons

In response to glutamatergic synaptic drive, striatal medium spiny neurons in vivo transition to a depolarized “up state” near spike threshold. In the up state, medium spiny neurons either depolarize enough to spike or remain below spike threshold and are silent before returning to the hyperpolarized “down state.” Previous work has suggested that subthreshold K+ channel currents were responsible for this dichotomous behavior, but the channels giving rise to the current and the factors determining its engagement have been a mystery. To move toward resolution of these questions, perforated-patch recordings from medium spiny neurons in tissue slices were performed. K+ channels with pharmacological and kinetic features of KCNQ channels potently regulated spiking at up-state potentials. Single-cell reverse transcriptase-PCR confirmed the expression of KCNQ2, KCNQ3, and KCNQ5 mRNAs in medium spiny neurons. KCNQ channel currents in these cells were potently reduced by M1 muscarinic receptors, because the effects of carbachol were blocked by M1 receptor antagonists and lost in neurons lacking M1 receptors. Reversal of the modulation was blocked by a phosphoinositol 4-kinase inhibitor, indicating a requirement for phosphotidylinositol 4,5-bisphosphate resynthesis for recovery. Inhibition of protein kinase C reduced the efficacy of the muscarinic modulation. Finally, acceleration of cholinergic interneuron spiking with 4-aminopyridine mimicked the effects of exogenous agonist application. Together, these results show that KCNQ channels are potent regulators of the excitability of medium spiny neurons at up-state potentials, and they are modulated by intrastriatal cholinergic interneurons, providing a mechanistic explanation for variability in spiking during up states seen in vivo.

Role of specific muscarinic receptor subtypes in cholinergic parasympathomimetic responses, in vivo phosphoinositide hydrolysis, and pilocarpine-induced seizure activity

Muscarinic agonist‐induced parasympathomimetic effects, in vivo phosphoinositide hydrolysis and seizures were evaluated in wild‐type and muscarinic M1–M5 receptor knockout mice. The muscarinic agonist oxotremorine induced marked hypothermia in all the knockout mice, but the hypothermia was reduced in M2 and to a lesser extent in M3 knockout mice. Oxotremorine‐induced tremor was abolished only in the M2 knockout mice. Muscarinic agonist‐induced salivation was reduced to the greatest extent in M3 knockout mice, to a lesser degree in M1 and M4 knockout mice, and was not altered in M2 and M5 knockout mice. Pupil diameter under basal conditions was increased only in the M3 knockout mice. Pilocarpine‐induced increases in in vivo phosphoinositide hydrolysis were completely absent in hippocampus and cortex of M1 knockout mice, but in vivo phosphoinositide hydrolysis was unaltered in the M2–M5 knockout mice. A high dose of pilocarpine (300u2003mg/kg) caused seizures and lethality in wild‐type and M2–M5 knockout mice, but produced neither effect in the M1 knockout mice. These data demonstrate a major role for M2 and M3 muscarinic receptor subtypes in mediating parasympathomimetic effects. Muscarinic M1 receptors activate phosphoinositide hydrolysis in cortex and hippocampus of mice, consistent with the role of M1 receptors in cognition. Muscarinic M1 receptors appear to be the only muscarinic receptor subtype mediating seizures.

The comparative geochemistries of lignins and carbohydrates in an anoxic fjord

Abstract A reducing, varved sediment core and monthly (May-September) plankton and sediment trap samples from Saanich Inlet, B.C., Canada, were analyzed for their elemental, lignin and neutral sugar compositions. Total yields of lignin-derived phenols from both the sediment trap and core samples indicated less than 15% and 30%, respectively, of chemically recognizable vascular plant remains, derived predominantly from gymnosperm wood and nonwoody angiosperm tissues. The elevated vanillyl and syringyl acid/aldehyde ratios of this material compared to fresh plant material indicated that it suffered mild aerobic decomposition prior to introduction to the Inlet. Most of the remaining particular organic material was nitrogen-rich, carbohydrate-poor and apparently plankton-derived. Organic carbon, total nitrogen, and total neutral sugars and lignin phenols all exhibited decreasing concentrations with depth in a region of uniform varving (upper 15 cm) in the sediment core. All profiles exhibited particularly steep concentration decreases within the top 2 cm of sediment. First-order decay constants for all four chemical categories within the upper 14 cm of the core ranged between 0.1–0.2 yr−1. Neutral sugars were consistently the most reactive chemical class, accounting for roughly 15% of the total organic carbon turnover. Although lignin appeared to be degraded within the sediment core, this degradation was nonselective for different lignin types and did not lead to increased acid/aldehyde ratios as occur during aerobic lignin decomposition. Comparisons of the yields of individual neutral sugars from the sediment and sediment trap samples to those expected from the vascular plant component alone indicated that the vascular plant debris in the upper portion of the sediment core had lost a portion of its initial glucose, lyxose, and mannose. In contrast, rhamnose and fucose were produced by all samples in large excess of total yields expected for chemically intact vascular plant and plankton components and must have additional sources.

Elucidating the role of muscarinic receptors in psychosis.

Muscarinic receptors have been implicated in the regulation of cognition and psychosis based on pharmacological evidence from pre-clinical and clinical studies. Muscarinic agonists have shown promise in the clinic in improving cognition and reducing psychotic episodes in Alzheimers patients. However, lack of selective muscarinic ligands has limited their use due to troublesome side effects observed at higher doses. Without selective ligands, it has been difficult to assign a specific muscarinic receptor subtype to these high order mental processes. Recent development of muscarinic receptor knockout mice has provided additional tools to investigate cognition and psychosis in behavioral assays and to determine the receptor subtypes associated with parasympathomimetic physiology. Biochemical studies indicate that the M1 receptor plays a significant role in regulating G alpha q-mediated signal transduction in the hippocampus and cortex. Behavioral studies suggest that the M4 receptor is involved in movement regulation and prepulse inhibition of the startle reflex, a measure of attention. These findings support a role for the development of M1 and M4 receptor agonists for diseases in which symptoms include cognitive impairment and psychotic behaviors.

M1 muscarinic acetylcholine receptors activate extracellular signal-regulated kinase in CA1 pyramidal neurons in mouse hippocampal slices.

Activation of extracellular signal-regulated kinases (ERK) is crucial for many neural functions, including learning, memory, and synaptic plasticity. As muscarinic acetylcholine receptors (mAChR) modulate many of the same higher brain functions as ERK, we examined mAChR-mediated ERK activation in mouse hippocampal slices. The cholinergic agonist carbachol caused an atropine-sensitive ERK activation in the dendrites and somata CA1 pyramidal neurons. To determine the responsible mAChR subtype, we combined pharmacologic and genetic approaches. Pretreatment with M1 antagonists inhibited ERK activation. Furthermore, mAChR-induced ERK activation was absent in slices from M1 knockout mice. ERK activation was normal in slices derived from other mAChR subtype knockouts (M2, M3, and M4), although these other subtypes are expressed in many of the same neurons. Thus, we demonstrate divergent functions for the different mAChR subtypes. We conclude that M1 is responsible for mAChR-mediated ERK activation, providing a mechanism by which M1 may modulate learning and memory.

M1 muscarinic receptor signaling in mouse hippocampus and cortex.

The five subtypes (M1-M5) of muscarinic acetylcholine receptors signal through G(alpha)(q) or G(alpha)(i)/G(alpha)(o). M1, M3 and M5 receptors couple through G(alpha)(q) and function predominantly as postsynaptic receptors in the central nervous system. M1 and M3 receptors are localized to brain regions involved in cognition, such as hippocampus and cortex, but their relative contribution to function has been difficult to ascertain due to the lack of subtype specific ligands. A functional and genetic approach was used to identify the predominant muscarinic receptor subtype(s) mediating responses in mouse hippocampus and cortex, as well as the relative degree of spare muscarinic receptors in hippocampus. The nonselective muscarinic agonist oxotremorine-M stimulated G(alpha)(q)/11-specific GTP-gamma-35S binding in a concentration dependent manner with a Hill slope near unity in wild type mouse hippocampus and cortex. Muscarinic receptor stimulated G(alpha)(q)/11-specific GTP-gamma-35S binding was virtually abolished in both the hippocampus and cortex of M1 receptor knockout (KO) mice. In contrast, there was no loss of signaling in M3 receptor KO mice in either brain region. Muscarinic receptor reserve in wildtype mouse hippocampus was measured by Furchgott analysis after partial receptor alkylation with propylbenzylcholine mustard. Occupation of just 15% of the M1 receptors in mouse hippocampus was required for maximal efficacy of oxotremorine-M-stimulated GTP-gamma-35S binding indicating a substantial level of spare receptors. These findings support a role for the M1 receptor subtype as the primary G(alpha)(q)/11-coupled muscarinic receptor in mouse hippocampus and cortex.

Identification of subtypes of muscarinic receptors that regulate Ca2+ and K+ channel activity in sympathetic neurons.

Many different G protein-coupled receptors modulate the activity of Ca2+ and K+ channels in a variety of neuronal types. There are five known subtypes (M1-M5) of muscarinic acetylcholine receptors. Knockout mice lacking the M1, M2, or M4 subtypes are studied to determine which receptors mediate modulation of voltage-gated Ca2+ channels in mouse sympathetic neurons. In these cells, muscarinic agonists modulate N- and L-type Ca2+ channels and the M-type K+ channel through two distinct, G-protein mediated pathways. The fast and voltage-dependent pathway is lacking in the M2 receptor knockout mice. The slow and voltage-independent pathway is absent in the M1 receptor knockout mice. Neither pathway is affected in the M4 receptor knockout mice. Muscarinic modulation of the M current is absent in the M1 receptor knockout mice, and can be reconstituted in a heterologous expression system using cloned channels and M1 receptors. Our results using knockout mice are compared with pharmacological data in the rat.

Muscarinic-induced modulation of potassium conductances is unchanged in mouse hippocampal pyramidal cells that lack functional M1 receptors

Activation of muscarinic acetylcholine (ACh) receptors (mAChRs) increases excitability of pyramidal cells by inhibiting several K+ conductances, including the after-hyperpolarization current (Iahp), the M-current (Im), and a leak K+ conductance (Ileak). Based on pharmacological evidence and the abundant localization of M1 receptors in pyramidal cells, it has been assumed that the M1 receptor is responsible for mediating these effects. However, given the poor selectivity of the pharmacological agents used to characterize these mAChR responses, rigorous characterization of the receptor subtypes that mediate these actions has not been possible. Surprisingly, patch clamp recording from CA1 pyramidal cells in M1 knockout mice revealed no significant difference in the degree of inhibition of Iahp, Im, or Ileak by the mAChR agonist, carbachol (CCh), as compared with wildtype controls. In addition, the M1-toxin was not able to block CChs inhibition of the Iahp, Im, or Ileak These data demonstrate that the M1 receptor is not involved in increasing CA1 pyramidal cell excitability by mediating ACh effects on these K+ conductances.

Role of the M1 receptor in regulating circadian rhythms.

Cholinergic stimuli are potent regulators of the circadian clock in the hypothalamic suprachiasmatic nucleus (SCN). Using a brain slice model, we have found that the SCN clock is subject to muscarinic regulation, a sensitivity expressed only during the night of the clocks 24-h cycle. Pharmacological and signal transduction characteristics are compatible with a response mediated by an M1-like receptor. Molecular manipulation of muscarinic receptors will provide important insights as to the receptor subtype(s) regulating circadian rhythms.