Histaminergic Modulation Of Neuronal Activity And Spatial Learning And Memory In Rat Entorhinal Cortex

Spatial learning and memory is one of the most important higher brain functions. Several lines of evidence indicate that entorhinal cortex(EC) plays a crucia l role in spatial learning and memory. Anatomically, EC is regarded as the “hub” of cortico-hippocampal circuits, and controls the flow of information into and out of the hippocampus. In addition, recent studies found that EC contains the spatial information-related functional cells such as grid cells, border cells, and actively participates in the spatial memory encoding and navigation. EC-related spatial cognition depends on the brain arousal state, the maintenance of which requires the histaminergic system. Several studies found that histamine alerts the neocortex via direct and indirect pathways, thus facilitating the spatial memory encoding. In hippocampus histamine excites the pyramidal neurons in the CA1 and granule cells in the DG, regulates the theta oscillations of the neuronal population, and increases the acetylcholine level via stimulating the acetylcholinergic neurons in the medial septum, consequently facilitating the hippocampal long-term potential and participating in the spatial cognition process.Since histaminergic system influences the neuronal activity and physiological functions of neocortex and hippocampus, we wonder whether the EC, which is the gateway between the neocortex and hippocampus, can be modulated by histamine. Although the E C receives histaminergic innervation, the function of histamine in the EC remains elusive. Therefore, in the present study, we used a combination of electrophysiology, behavioral pharmacology, and immunohistochemical approaches to clarify the functions of histamine in the EC at the molecular, cellular and behavioral levels.In addition, we also note that orexins are involved in the initiation and maintenance of the brain arousal state. Recently, orexins have been reported to regulate neuronal activity and synaptic transmission in the hippocampus and prefrontal cortex, and participate in the rat spatial learning. Since the EC is highly related to spatial cognition, we wonder whether orexin system modulates neuronal activity in the EC. Therefore, in this study, we also observed the neuromodulatory effects of orexins in the superficial layers of the EC.The main results are as follows:1. Histamine and Orexin Systems Increase the Excitability of Principal Neurons in the Superficial of the ECStellate and pyramidal neurons are the principal projection neurons in the superficial layers of the EC. These two types of neurons can be identified by their shape and electrophysiological properties. Histamine(1-1000 μM) reversibly depolarized these two types of neurons in a concentration-dependent manner, and in some occasions stimulated the neuronal firing. Also, under voltage-clamp model, histamine can induce an inward current(n = 5, P < 0.01). Nevertheless, in the deep layers of the EC histamine(10-1000 μM) did not affect the membrane potential(10 μM: n = 8; P = 0.61; 100 μM: n = 10, P = 0.22; 1 m M: n = 10, P = 0.17) and whole-cell currents(10 μM: n = 19; P = 0.94; 100 μM: n = 12, P = 0.07; 1 m M: n = 13, P = 0.19), indicating that histamine specifically excites the p rincipal neurons in the superficial layers of the EC.Accordingly, we also investigated the effects of orexin system, another important arousal-promoting system, on the projection neurons of the superficial layers of the EC. Orexin-A at 100 n M induced depolarization(n = 17; P < 0.001) of membrane potentials and inward current(n = 5; P < 0.01) for the stellate neurons in the superficial layers of the EC. Also, orexin-A depolarized the membrane potentials of the pyramidal neurons in layer II(n = 9, P < 0.001) and III of EC(n = 6, P < 0.001). Orexin-A-induced depolarization was not significantly different among these three types of neurons(H2 = 0.24; P = 0.89). These results suggest that orexins also excite the projection neurons in the superficial layers of the EC in a cell type-nonspecific manner.2. The Postsynaptic Effect of Histamine Is Mediated by H1Rs-PKC Pathway Dependent Inhibition of Kir ChannelsIn the presence of Trip, a selective H1 R antagonist, the histamine-elicited depolarization was abolished(n = 5, P < 0.05). However, histamine-induced depolarization was not affected by the H2 R blocker Rani(n = 8, P = 0.32) or H3 R blocker Clob(n = 4, P = 0.59). The results of immunofluorescence revealed that H1 Rs were mainly expressed in the superficial layers, but not in deep layers, of the EC. While, the distribution of H 2Rs in the superficial layers and deep layers of the EC was relative scarce. After bath incubation with PKC inhibitor BIS-II at least for 90 min, the effect of histamine in the EC was also blocked(n = 10; P < 0.001). Together, these results suggest that histamine-induced excitation in the superficial layers is mediated by activation of H1Rs-PKC pathway.We also tested whether the histamine-induced depolarization was a direct postsynaptic effect. Histamine-induced depolarization was persisted in the presence of either TTX(n = 5, P = 0.81) or CNQX, AP5 and PIC(n = 5, P = 0.38) known to block inhibitory and excitatory transmission. Moreover, in the presence of histamine, the input conductance of the cells was slightly decreased(n = 4, P < 0.05), indicating a postsynaptic action.The slow ramp command test(d V/dt =-10 m V/s) was conducted to obtain histamine-induced difference currents. In an ACSF with Ko at 3 m M, a reversal of the histamine effect was –87 ± 1 m V, thus very close to the estimated Ek. Performing the same experiment in an ACSF with Ko at 10 m M, a reversal for the histamine effect was-59 ± 1 m V, thus again very close to the estimated Ek. Next, we applied the classic K+ channel blockers. Histamine-induced depolarization was blocked Ba2+(n = 6, P < 0.01) or Cs+(n = 5, P < 0.01), while this effect was insensitive to the voltage-gated potassium channel blocker TEA(n = 4, P = 0.89) or 4AP(n = 6, P = 0.77). These findings indicate that the direct effect of histamine is mediated by inhibition of inwardly recitifying potassium channels.3. Histamine Selectively Decreases Spontaneous GABA Release in the Superficial, but Not Deep, Layers of the ECUsing whole-cell patch-clamp combined pharmacological approach, we isolated the miniature excitatory postsynaptic currents(m EPSCs) and miniature inhibitory postsynaptic currents(m IPSCs) in the EC. In the superficial layers of the EC, histamine reduced the frequency of m IPSCs(n = 7, P < 0.001), while it did not affect the m IPSC amplitude(n = 7, P = 0.62). The histamine did not influence the frequency and amplitude of m EPSCs in the superficial layers. In the deep layers, both the m IPSCs and m EPSCs were not affected by the histamine, indicating that histamine specifically inhibits the presynaptic GABA release in the superficial layers of the EC.4. Inhibition of Spontaneous GABA Release by Histamine in the Superficial Layers Requires H3Rs-Dependent Modulation of the VGCCsWe added histamine receptor antagonists into the ACSF and explored their effects on the histamine-induced inhibition of GABA release. Neither Trip nor Rani changed the histamine-induced reduction of the frequency of m IPSCs(Trip: n = 6, P < 0.05; Rani: n = 7, P < 0.05). However, histamine-mediated decrease in the frequency of m IPSCs was blocked by H3 R antagonist(n = 6, P = 0.68). In the superficial layers, morphological results showed that H3 Rs were always expressed on the inhibitory synaptic terminals. Instead, the expression level of these receptors in the deep layers was relatively low. These results suggest that the presynaptic effect of histamine is mediated by presynaptic H 3Rs.Bath application of Cd2+, a general VGCC blocker, alone induced a rightward shift of the interevent interval distribution of m IPSCs(P < 0.001, K-S test) and decreased the frequency to 57 ± 6% of control(n = 7, P < 0.01). After pretreatment with Cd2+, histamine failed to alter the frequency of m IPSCs(n = 7, P = 0.80). Also, histamine did not induce a significant change in the frequency of m IPSCs in Ca2+-free external solution(n = 5, P = 0.65). These results suggest that H3Rs-induced inhibition of GABA release depends on extracellular Ca2+ passing through the VGCCs.5. Blockade of H1 Rs or H3 Rs in the Superficial Layers of the EC Impairs Rat Spatial Learning without Effects on Rat Locomotor and Exploratory ActivitiesWe next explored the effects of histamine in the EC on rat spatial learning in the Morris water maze task. The rats receiving intra-superficial layer infusion of H1 R or H3 R antagonist had longer escape latencies than the ACSF-treated group(Trip: P < 0.001; Clob: P < 0.01). Conversely, microinjection of the H2 R antagonist failed to alter the escape latencies compared to the ACSF-treated group(P = 0.86). Nonspatial learning factors, such as the motivational, emotional and motor functions of the tested subjects, have been reported to influence the water maze performance. Thus we analyzed the recorded swimming speed of the rats. Intra-superficial layer application of the drugs did not alter the swimming speed of the rats(F4, 57 = 0.99; P = 0.42). Also, in the open field test, both the spontaneous locomotor(F4, 33 = 0.59; P = 0.68) and exploratory activity(F4, 33 = 0.23; P = 0.92) of rats were not affected by microinjecting of drugs. These results demonstrate that histamine in the superficial layers of the EC specifically affects the spatial learning rather than a secondary effect of motivational, emotional or motor functions.6. Blockade of Histamine Receptors in the Deep Layers of the EC does not Affect the Rat Spatial Learning, Locomotor and Exploratory Activities.We also microinjected histamine receptor antagonists into the deep layers of the EC and investigated their effects on rat spatial learning. There was no overall group difference in escape latencies to find the hidden platform(F4, 44 = 1.19; P = 0.33) and no groups-by-trials interaction(F44, 484 = 0.60; P = 0.98), reflecting the equivalent improvement of both groups across the acquisition stage. Similarly, intra-deep layer infusion of the drugs did not affect the swimming speed of the rats(F4, 44 = 1.38; P = 0.26). Also, the spontaneous locomotor(F4, 34 = 0.27; P = 0.90) and exploratory activity(F4, 34 = 0.10; P = 0.98) of rats in an open-field were not affected by microinjecting of the above drugs. These findings indicate that the blockade of the histamine receptors in the deep layers of the EC does not affect the rat learning ability.In summary, these results suggest that histamine and orexins excite the projection neurons in superficial layers of EC. The neuromodulatory effects of histamine are mediated via inhibition of postsynaptic inhibition of inwardly recitifying potassium channels and presynaptic GABA release, and contribute to the superficial layers of EC-related spatial cognition. These findings provide a novel neural mechanism to explain how the arousal system modulates cognition. Moreover, they provide a new insight for the treatme nt of cognitive dysfunctions.