Selective wheat germ agglutinin (WGA) uptake in the hippocampus from the locus coeruleus of dopamine-β-hydroxylase-WGA transgenic mice

We generated transgenic mice in which a trans-synaptic tracer, wheat germ agglutinin (WGA), was specifically expressed in the locus coeruleus (LC) neurons under the control of the dopamine-β-hydroxylase (DBH) gene promoter. WGA protein was produced in more than 95% of the tyrosine hydroxylase (TH)-positive LC neurons sampled. Transynaptic transfer of WGA was most evident in CA3 neurons of the hippocampus, but appeared absent in CA1 neurons. Faint but significant WGA immunoreactivity was observed surrounding the nuclei of dentate granule cells. Putative hilar mossy cells, identified by the presence of calretinin in the ventral hippocampus, appeared uniformly positive for transynaptically transferred WGA protein. GAD67-positive interneurons in the hilar and CA3 regions tended to be WGA-positive, although a subset of them did not show WGA co-localization. The same mixed WGA uptake profile was apparent when examining co-localization with parvalbumin. The selective uptake of WGA by dentate granule cells, mossy cells, and CA3 pyramidal neurons is consistent with evidence for a large proportion of conventional synapses adjacent to LC axonal varicosities in these regions. The lack of WGA uptake in the CA1 region and its relatively sparse innervation by DBH-positive fibers suggest that a majority of the TH-positive classical synapses revealed by electron microscopy in that region may be producing dopamine. The overall pattern of WGA uptake in these transgenic mice implies a selective role for the granule cell-mossy cell-CA3 network in processing novelty or the salient environmental contingency changes signaled by LC activity

Locus Coeruleus Optogenetic Light Activation Induces Long-Term Potentiation of Perforant Path Population Spike Amplitude in Rat Dentate Gyrus

Norepinephrine (NE) in dentate gyrus (DG) produces NE-dependent long-term potentiation (NE-LTP) of the perforant path-evoked potential population spike both in vitro and in vivo. Chemical activators infused near locus coeruleus (LC), the source of DG NE, produce a NE-LTP that is associative, i.e., requires concurrent pairing with perforant path (PP) input. Here, we ask if LC optogenetic stimulation that allows us to activate only LC neurons can induce NE-LTP in DG. We use an adeno-associated viral vector containing a depolarizing channel (AAV8-Ef1a-DIO-eChR2(h134r)-EYFP-WPRE) infused stereotaxically into the LC of TH:Cre rats to produce light-sensitive LC neurons. A co-localization of ~62% in LC neurons was observed for these channels. Under urethane anesthesia, we demonstrated that 5–10 s 10 Hz trains of 30 ms light pulses in LC reliably activated neurons near an LC optoprobe. Ten minutes of the same train paired with 0.1 Hz PP electrical stimulation produced a delayed NE-LTP of population spike amplitude, but not EPSP slope. A leftward shift in the population spike input/output curve at the end of the experiment was also consistent with long-term population spike potentiation. LC neuron activity during the 10 min light train was unexpectedly transient. Increased LC neuronal firing was seen only for the first 2 min of the light train. NE-LTP was more delayed and less robust than reported with LC chemo-activation. Previous estimates of LC axonal conduction times suggest acute release of NE occurs 40–70 ms after an LC neuron action potential. We used single LC light pulses to examine acute effects of NE release and found potentiated population spike amplitude when a light pulse in LC occurred 40–50 ms, but not 20–30 ms, prior to a PP pulse, consistent with conduction estimates. These effects of LC optogenetic activation reinforce evidence for a continuum of NE potentiation effects in DG. The single pulse effects mirror an earlier report using LC electrical stimulation. These acute effects support an attentional role of LC activation. The LTP of PP responses induced by optogenetic LC activation is consistent with the role of LC in long-term learning and memory

Locus Ceruleus Activation Initiates Delayed Synaptic Potentiation of Perforant Path Input to the Dentate Gyrus in Awake Rats: A Novel �-Adrenergic- and Protein Synthesis- Dependent Mammalian Plasticity Mechanism

Norepinephrine, acting through β-adrenergic receptors, is implicated in mammalian memory. In in vitro and in vivo studies, norepinephrine produces potentiation of the perforant path-dentate gyrus evoked potential; however, the duration and dynamics of norepinephrine-induced potentiation have not been explored over extended time periods. To characterize the long-term effects of norepinephrine on granule cell plasticity, the present study uses glutamatergic activation of the locus ceruleus (LC) to induce release of norepinephrine in the hippocampus of the awake rat and examines the subsequent modulation of the dentate gyrus evoked potential for 3 hr (short term) and 24 hr (long term) after LC activation. LC activation initiates a potentiation of the field EPSP slope observed 24 hr later. This late-phase potentiation of the synaptic potential is not preceded by early phase potentiation, although spike potentiation can be seen both immediately after, and 24 hr after, LC activation. Intracerebroventricular infusion of the β-adrenergic antagonist, propranolol, or the protein synthesis inhibitor, anisomycin, before LC activation blocks the potentiation of perforant path input observed at 24 hr. The initiation of late-phase synaptic potentiation observed at 24 hr but not at the 3 hr after LC activation parallels the observation of a cAMP- and protein synthesis-dependent long-lasting synaptic facilitation in Aplysia that is not preceded by short-term synaptic facilitation. Locus ceruleus-initiated synaptic potentiation may selectively support long-term, rather than short-term, memory. The observation of selective initiation of long-term synaptic facilitation in a mammalian brain, as in invertebrates, is additional evidence that these two forms of memory depend on separable biological mechanisms

Locus ceruleus activation suppresses feedforward interneurons and reduces β-γ electroencephalogram frequencies while it enhances θ frequencies in rat dentate gyrus

The locus ceruleus is activated by novel stimuli, and its activation promotes learning and memory. Phasic activation of locus ceruleus neurons by glutamate enhances the dentate gyrus population spike amplitude and results in long-term potentiation of synaptic responses recorded after 24 h. Cholinergic activation of locus ceruleus neurons increases hippocampal θ. At the level of the cellular network, it is not clear how the potentiating effects of norepinephrine are mediated. Previous studies show that exogenous norepinephrine enhances inhibitory interneuron firing in the dentate gyrus. This finding appears at odds with evidence for potentiation. In this study, natural release of norepinephrine was induced by glutamate activation of locus ceruleus while we recorded EEGs and physiologically identified interneurons in the dentate gyrus of urethane-anesthetized rats. Feedforward neurons were inhibited (∼1-2 min) by locus ceruleus activation. Feedback interneurons showed both increased and decreased activity, whereas granule cells increased firing as predicted by evoked potential studies. EEG results replicated an increase in θ power (4-8 Hz) with locus ceruleus activation, but the effect with glutamatergic locus ceruleus activation was transient (∼1-2 min). β-γ Frequencies were also transiently suppressed. Together, the data suggest that locus ceruleus activation enhances the throughput of concomitant sensory input by reducing feedforward inhibitory interneuron activity, which may reduce "binding" in existing cell assemblies, and enhances the conditions for synaptic plasticity through disinhibition, promotion of 4-8 Hz θ, and noradrenergic potentiation to facilitate the building of new representations

Orexin-A Infusion in the Locus Ceruleus Triggers Norepinephrine (NE) Release and NE-Induced Long-Term Potentiation in the Dentate Gyrus

The orexins (ORX-A/ORX-B) are neuroactive peptides known to have roles in feeding and sleep. Evidence of dense, excitatory projections of ORX-A neurons to the noradrenergic pontine nucleus, the locus ceruleus (LC), suggests ORX-A also participates in attention and memory. Activation of LC neurons by glutamate produces a β-adrenergic receptor-mediated long-term potentiation (LTP) of the perforant path-evoked potential in the dentate gyrus, a target structure of the LC that has been implicated in memory. We asked whether ORX-A also activates norepinephrine (NE)-induced LTP by initiating NE release in the hippocampus. Here, we show that ORX-A infusion (0.25-25 fmol) into the LC produces a robust, β-adrenergic receptor-dependent, long-lasting potentiation of the perforant path-evoked dentate gyrus population spike in the anesthetized rat. Pharmacological inactivation of the LC with an α2-adrenergic receptor agonist, before ORX-A infusion, prevents this potentiation. Analysis of NE concentrations in the hippocampus after ORX-A infusion into the LC reveals a transient, but robust, increase in NE release. Thus, this study demonstrates that the dense orexinergic projection to the LC promotes the induction of NE-LTP in the dentate gyrus. ORX-A modulation of LC activity may provide important support for the cognitive processes of attention and memory

Insights into the effects of norepinephrine on memory: studies of noradrenergic modulation of synaptic plasticity in the dentate gyrus of the rat

Norepinephrine (NE) is known to increase memory for emotional events. This catacholamine, applied exogenously or through natural release mechanisms, increases memory in rats and humans, and increases cell excitability in areas of the rat brain (NE-induced potentiation), in a manner congruent with other models of long-lasting memory support. The purpose of this dissertation is to investigate the effects of NE on synaptic efficacy in rat dentate gyrus, a component of the memory structure, the hippocampus. Three chapters are presented, each utilizing a different method of intensifying the synaptic levels of NE in the dentate gyrus, and investigating the ensuing effects on the perforant path-dentate gyrus evoked potential. -- In the first chapter, exogenous NE and noradrenergic agents were applied in the lateral ventricle of the awake rat, a technique used to meld in vitro bath application of drugs, with in vivo whole animal recording in the absence of anesthetic. Here it was found that NE, infused ventricularly reliably increased the synaptic contribution of the evoked potential (EPSP slope), a result characteristic of bath application of NE in the hippocampal slice. NE also increased dentate granule cell firing as indexed by the population spike amplitude of the evoked response. Though these initial increases returned to baseline within 30 min, a subset of animals monitored 24 hr after NE infusion, demonstrated long-term potentiation of the EPSP slope and population spike amplitude. Both the short-term and long-term potentiation were dependent on β-adrenergic receptor activation. The data suggest NE can mediate multiple phases of synaptic plasticity and long-term potentiation may not reflect an uninterrupted progression from short-term potentiation, a widely held theory of how long-term memories are formed. -- In the second chapter, the evoked population activity in the dentate gyrus of the anesthetized rat was monitored while the recently discovered neuropeptide orexin-A (OREX-A) was infused directly into the LC as a method of discretely activating LC neurons to evoke NE release in the hippocampus. Application of OREX-A at 3 concentrations (1, 10, and 100 nM) produced a robust potentiation of the population spike amplitude lasting for greater than 3 hr. This potentiation was reduced by intradentate application of the β-adrenergic receptor antagonist propranolol. Infusion of vehicle into the LC failed to produce changes in the evoked activity. LC infusion of the α₂₋adrenergic receptor agonist clonidine prior to OREX-A infusion, a method of pharmacologically inactivating LC neurons, blocked the effect of OREX-A. Lastly, hippocampal levels of NE were monitored to confirm that infusion of OREX-A into the LC produced a transient elevation of NE levels in the hippocampus. This is the first study to investigate hippocampal synaptic effects of orexinergic activation of LC neurons and the first to suggest that OREX-A can produce changes in synaptic activity reminiscent of memory formation. -- The third study takes advantage of a technique developed in the anesthetized preparation whereby endogenous release of NE is initiated by the application of the excitatory amino acid transmitter glutamate to the noradrenergic neurons of the LC. As anesthetized preparations are limited by the duration over which effects can be monitored, this technique was used in the awake rat to assess the effects of NE on the dentate gyrus evoked potential at periods 24 hr after LC activation. Glutamatergic activation of the LC neurons produced a robust facilitation of the dentate gyrus population spike and EPSP slope, an increase more sizable than that seen within the first 3 hr following LC activation. This effect is unusual in that short-term potentiation of the evoked potential was not a necessary requirement for the NE-induced long-term potentiation observed at 24 hr. Though this effect is novel in the mammalian nervous system, there are behavioral studies that are in keeping with this finding and similar synaptic effects have been seen in an invertebrate. These effects were dependent on the activation of β-adrenergic receptors and on the synthesis of de novo protein. -- Taken together these studies suggest locus coeruleus activation has a special role in the initiation of long-term increases in synaptic strength and cell responses in circuitry known to be critical for memory formation and, further, that short-term and long-term synaptic plasticity may be supported by distinct and independent processes

Sprague-Dawley Rats Differ in Responses to Medial Perforant Path Paired Pulse and Tetanic Activation as a Function of Sex and Age

Network plasticity in the medial perforant path (MPP) of adult (five to nine months) and aged (18–20 months) urethane-anesthetized male and female Sprague Dawley rats was characterized. Paired pulses probed recurrent networks before and after a moderate tetanic protocol. Adult females exhibited greater EPSP-spike coupling suggesting greater intrinsic excitability than adult males. Aged rats did not differ in EPSP-spike coupling but aged females had larger spikes at high currents than males. Paired pulses suggested lower GABA-B inhibition in females. Absolute population spike (PS) measures were larger post-tetani in female rats than male rats. Relative population spike increases were greatest in adult males relative to females and to aged males. EPSP slope potentiation was detected with normalization in some post-tetanic intervals for all groups except aged males. Tetani shortened spike latency across groups. Tetani-associated NMDA-mediated burst depolarizations were larger for the first two trains in each tetanus in adult males than other groups. EPSP slopes over 30 min post-tetani predicted spike size in female rats but not in males. Replicating newer evidence MPP plasticity in adult males was mediated by increased intrinsic excitability. Female MPP plasticity was related to synaptic drive increases, not excitability increases. Aged male rats were deficient in MPP plasticity