Seasonal variation of long-term potentiation at a central synapse in the medicinal leech.

Long-term potentiation (LTP) is a persistent increase in synaptic transmission that is thought to contribute to a variety of adaptive processes including learning and memory. Although learning is known to undergo circannual variations, it is not known whether LTP undergoes similar changes despite the importance of LTP in learning and memory. Here we report that synapses in the CNS of the medicinal leech demonstrate seasonal variation in the capacity to undergo LTP following paired presynaptic and postsynaptic stimulation. LTP was observed during the April-October period, but no LTP was observed during the November-March period. Application of forskolin, a technique often used to produce chemical LTP, failed to elicit potentiation during the November-March period. Implementing stimulation patterns that normally result in long term depression (LTD) also failed to elicit any change in synaptic strength during the November-March period. These experiments indicate that LTP and LTD can be influenced by circannual rhythms and also suggest a seasonal influence on learning and memory.

Co-induction of LTP and LTD and its regulation by protein kinases and phosphatases.

The cellular properties of long-term potentiation (LTP) following pairing of pre- and postsynaptic activity were examined at a known glutamatergic synapse in the leech, specifically between the pressure (P) mechanosensory and anterior pagoda (AP) neurons. Stimulation of the presynaptic P cell (25 Hz) concurrent with a 2 nA depolarization of the postsynaptic AP cell significantly potentiated the P-to-AP excitatory postsynaptic potential (EPSP) in an N-methyl-d-aspartate receptor (NMDAR)-dependent manner based on inhibitory effects of the NMDAR antagonist MK801 and inhibition of the NMDAR glycine binding site by 7-chlorokynurenic acid. LTP was blocked by injection of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) into the postsynaptic (AP) cell, indicating a requirement for postsynaptic elevation of intracellular Ca(2+). Autocamtide-2-related inhibitory peptide (AIP), a specific inhibitor of Ca(2+)/calmodulin-dependent kinase II (CaMKII), and Rp-cAMP, an inhibitor of protein kinase A (PKA), also blocked pairing-induced potentiation, indicating a requirement for activation of CaMKII and PKA. Interestingly, application of AIP during pairing resulted in significantly depressed synaptic transmission. Co-application of AIP with the protein phosphatase inhibitor okadaic acid restored synaptic transmission to baseline levels, suggesting an interaction between CaMKII and protein phosphatases during induction of activity-dependent synaptic plasticity. When postsynaptic activity preceded presynaptic activity, NMDAR-dependent long-term depression (LTD) was observed that was blocked by okadaic acid. Postsynaptic injection of botulinum toxin blocked P-to-AP potentiation while postsynaptic injection of pep2-SVKI, an inhibitor of AMPA receptor endocytosis, inhibited LTD, supporting the hypothesis that glutamate receptor trafficking contributes to both LTP and LTD at the P-to-AP synapse in the leech.

Molecular identification and expression of the NMDA receptor NR1 subunit in the leech.

The N-methyl-D-aspartate receptor (NMDAR) is involved in a number of physiological and pathophysiological processes in vertebrates, but there have been few studies examining the role of invertebrate NMDA receptors. In the leech, pharmacological evidence suggests that NMDARs contribute to synaptic plasticity, but there has been no molecular identification of NMDA receptors. In this report, a partial cDNA encoding the leech NR1 subunit of the NMDA receptor (HirNR1) is presented. Reverse transcriptase-polymerase chain reaction from single neurons of the leech central nervous system confirms HirNR1 expression in the Retzius (R), Anterior Pagoda (AP), Pressure (P), and Touch (T) neurons. Immunoblotting with an anti-NR1 antibody yielded a approximately 110 kDa protein, similar to the expected weight of the NR1 subunit (approximately 116 kDa). Finally, pairing pre- and postsynaptic activity elicited long-term potentiation in synapses between neurons expressing NR1 mRNA (P-to-AP synapse) and this potentiation was blocked by the NMDAR antagonist AP5.

Forskolin induces NMDA receptor-dependent potentiation at a central synapse in the leech.

In vertebrate hippocampal neurons, application of forskolin (an adenylyl cyclase activator) and rolipram (a phosphodiesterase inhibitor) is an effective technique for inducing chemical long-term potentiation (cLTP) that is N-methyl-d-aspartate (NMDA) receptor (NMDAR)-dependent. However, it is not known whether forskolin induces a similar potentiation in invertebrate synapses. Therefore, we examined whether forskolin plus rolipram treatment could induce potentiation at a known glutamatergic synapse in the leech (Hirudo sp.), specifically between the pressure (P) mechanosensory and anterior pagoda (AP) neurons. Perfusion of isolated ganglia with forskolin (50 muM) in conjunction with rolipram (0.1 muM) in Mg(2+)-free saline significantly potentiated the P-to-AP excitatory postsynaptic potential. Application of 2-amino-5-phosphonovaleric acid (APV, 100 muM), a competitive NMDAR antagonist, blocked the potentiation, indicating P-to-AP potentiation is NMDAR-dependent. Potentiation was blocked by injection of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA, 1 mM) into the postsynaptic cell, but not by BAPTA injection into the presynaptic neuron, indicating a requirement for postsynaptic elevation of intracellular Ca(2+). Application of db-cAMP mimicked the potentiating effects of forskolin, and Rp-cAMP, an inhibitor of protein kinase A, blocked forskolin-induced potentiation. Potentiation was also blocked by autocamtide-2-related inhibitory peptide (AIP), indicating a requirement for activation of Ca(2+)/calmodulin-dependent kinase II (CaMKII). Finally, potentiation was blocked by botulinum toxin, suggesting that trafficking of glutamate receptors also plays a role in this form of synaptic plasticity. These experiments demonstrate that techniques used to induce cLTP in vertebrate synapses also induce NMDAR-dependent potentiation in the leech CNS and that many of the cellular processes that mediate LTP are conserved between vertebrate and invertebrate phyla.