Balanced anesthesia in pigeons (Columba livia): a protocol that ensures stable vital parameters and feasibility during long surgeries in cognitive neuroscience.

In neuroscience, numerous experimental procedures in animal models require surgical interventions, such as the implantation of recording electrodes or cannulas before main experiments. These surgeries can take several hours and should rely on principles that are common in the field of research and medicine. Considering the characteristics of the avian respiratory physiology, the development of a safe and replicable protocol for birds is necessary to minimize side effects of anesthetic agents, circumvent technical limitations due to the insufficient availability of patient monitoring, and to maintain stable intraoperative anesthesia. Through the consistent and responsible implementation of the three R principle of animal welfare in science ("Replace, Reduce, Refine"), we aimed to optimize experimental methods to minimize the burden on pigeons (Columba livia) during surgical procedures. Here, surgeries were conducted under balanced anesthesia and perioperative monitoring of heart rate, oxygen saturation, body temperature, and the reflex state. The protocol we developed is based on the combination of injectable and inhalative anesthetic drugs [ketamine, xylazine, and isoflurane, supported by the application of an opiate for analgesia (e.g., butorphanol, buprenorphine)]. The combination of ketamine and xylazine with a pain killer is established in veterinary medicine across a vast variety of species. Practicability was verified by survival of the animals, fast and smooth recovery quantified by clinical examination, sufficiency, and stability of anesthesia. Independent of painful stimuli like incision or drilling, or duration of surgery, vital parameters were within known physiological ranges for pigeons. Our approach provides a safe and conservative protocol for surgeries of extended duration for scientific applications as well as for veterinary medicine in pigeons which can be adapted to other bird species.

Regulation of long-term depression by increases in [guanosine 3',5'-cyclic monophosphate] in the hippocampal CA1 region of freely behaving rats.

A role for guanosine 3',5'-cyclic monophosphate (cGMP) and the protein kinase G (PKG) pathway in synaptic long-term depression (LTD) in the hippocampal CA1 region has been proposed, based on observations in vitro, where, for example, increases of [cGMP] result in short-term depression (STD) coupled with a reduction in presynaptic glutamate release. To date, no evidence exists to support that LTD in the intact, freely behaving animal involves these mechanisms. We examined the effect of increases of [cGMP] on basal transmission and electrically-induced STD at hippocampal CA1 synapses in vivo. We found that elevating [cGMP] dose-dependently caused a chemically-induced STD which occluded electrically-induced STD. Repeated administration of Zaprinast, an inhibitor of cGMP-degrading phosphodiesterase, resulted in persistent LTD (>24 h). Paired-pulse analysis supported a presynaptic mechanism of action. Application of an inhibitor of soluble guanylate cyclase prevented LTD induced by low-frequency stimulation (LFS), and impaired LFS-STD elicited in the presence of Zaprinast. These data suggest the involvement of cGMP in LTD in the CA1 region of freely behaving adult rats.

A specific role for group II metabotropic glutamate receptors in hippocampal long-term depression and spatial memory.

Activity-dependent and sustained alterations in synaptic efficacy are widely regarded as the cellular correlates underlying learning and memory. Metabotropic glutamate receptors (mGluRs) are intrinsically involved in both hippocampal synaptic plasticity and spatial learning. Group II mGluRs are required for persistent hippocampal long-term depression (LTD), but are not required for long-term potentiation (LTP) in the hippocampal CA1 region in vivo. The role of these receptors in spatial learning, and in synaptic plasticity in the dentate gyrus in vivo has not yet been the subject of close scrutiny. We investigated the effects of group II mGluR antagonism on LTP and LTD in the adult rat, at medial perforant path-dentate gyrus synapses, and on spatial learning in the eight-arm radial maze. Daily application of the group 2 mGluR antagonist (2S)-alpha-ethylglutamic acid (EGLU) resulted in impairment of long-term (reference) memory with effects becoming apparent 6 days after training and drug-treatment began. Short-term (working) memory was unaffected throughout the 10-day study. Acute injection of EGLU did not affect either LTD or LTP in the dentate gyrus in vivo. Following six daily applications of EGLU a clear impairment of LTD but not LTP was apparent however. These data support that prolonged antagonism of group II mGluRs results in an impairment of LTD that parallels the appearance of spatial memory deficits arising from group II mGluR antagonism. These findings support the importance of group II mGluRs for spatial memory formation and offer a further link between LTD and the encoding of spatial information in the hippocampus.

Requirement of translation but not transcription for the maintenance of long-term depression in the CA1 region of freely moving rats.

Hippocampal long-term depression (LTD) comprises a persistent reduction in synaptic strength that can be induced in the CA1 region by repeated low-frequency stimulation (LFS). Previous studies have demonstrated that hippocampal long-term potentiation requires de novo protein synthesis. Whether hippocampal LTD is also protein synthesis-dependent is not known. In this study, we investigated if the previous administration of translation inhibitors (anisomycin or emetine) or a transcription inhibitor (actinomycin-D) influenced the profile of LTD in freely moving adult Wistar rats. Seven- to 8-week-old animals underwent chronic implantation of a recording electrode in the CA1 stratum radiatum and a stimulation electrode in the Schaffer collateral/commissural fiber pathway. A cannula was implanted in the ipsilateral cerebral ventricle to enable drug administration. Experiments were commenced 10 d after the implantation procedure. Immediately after application of LFS (1 Hz, 900 pulses) robust LTD was seen that persisted for >8 hr in control animals. Application of anisomycin (240 microg/5 microl) emetine (240 microg/5 microl) before LFS prevented the expression of LTD or approximately 4.5 hr after LFS. Previous administration of actinomycin D (72 microg/12 microl) had no effect on the expression of LTD. None of the compounds elicited significant effects on basal synaptic transmission when administered in the absence of LFS. These data suggest that LTD in the CA1 region in vivo is protein synthesis-dependent. Furthermore, persistent LTD can be established through the translation of existing mRNA, whereas de novo mRNA transcription does not appear to be necessary.

Synaptic strength at the temporoammonic input to the hippocampal CA1 region in vivo is regulated by NMDA receptors, metabotropic glutamate receptors and voltage-gated calcium channels.

The hippocampal CA1 region receives cortical information via two main inputs: directly via the perforant (temporoammonic) path (pp-CA1 synapse) and indirectly via the tri-synaptic pathway. Although synaptic plasticity has been reported at the pp-CA1 synapse of freely behaving animals, the mechanisms underlying this phenomenon have not been investigated. Here, we explored whether long-term potentiation (LTP) at the pp-CA1 synapse in freely behaving rats requires activation of N-methyl-d-aspartate receptors (NMDAR) and L-type voltage-gated calcium channels (VGCCs). As group II metabotropic glutamate (mGlu) receptors are densely localized on presynaptic terminals of the perforant path, and are important for certain forms of hippocampal synaptic plasticity, we also explored whether group II mGlu receptors affect LTP at the pp-CA1 synapse and/or regulate basal synaptic transmission at this synapse in vivo. In adult male rats, high-frequency stimulation (200Hz) given as 3, or 10 trains, resulted in robust LTP that lasted for at least 4h in pp-CA1 or pp-dentate gyrus (DG) synapses, respectively. Pre-treatment with the NMDAR antagonist D-(-)-2-amino-5-phosphopentanoic acid (D-AP5) partially inhibited LTP at pp-CA1, and completely prevented LTP at pp-DG synapses. Combined antagonism of NMDAR using D-AP5 and the VGCC inhibitor, (-)-methoxyverapamil hydrochloride elicited a further inhibition of the LTP response at pp-CA1 synapses. Whereas activation of group II mGlu receptors using (1R,2R)-3-((1S)-1-amino-2-hydroxy-2-oxoethyl) cyclopropane-1,2-dicarboxylic acid (DCG-IV) dose-dependently reduced basal synaptic transmission elicited by test-pulse stimulation, DCG-IV did not affect LTP in a dose that inhibited LTP at pp-DG synapses in vivo. These data indicate that LTP at the pp-CA1 synapse of freely behaving animals is dually dependent on NMDAR and VGCCs, whereby group II mGlu receptor activation affect basal synaptic tonus, but not LTP. The lower frequency-dependency of NMDA-VGCC LTP at pp-CA1 synapses compared to pp-DG synapses may comprise a mechanism to prioritize information processing at this synapse.

Ras does not contribute to the facilitation of hippocampal synaptic plasticity enabled by environmental enrichment.

Environmental enrichment (EE), which mimics the wealth of sensory, motor and cognitive stimuli that arise through intense interactions with the ambient environment, results in enhanced hippocampal long-term potentiation (LTP) and spatial learning. A key molecular factor in the mediation of these changes is the brain-derived neurotrophic factor (BDNF). One of the downstream cascades that is activated by BDNF is the cascade linked to the small GTPase, Ras, that triggers mitogen-activated protein kinase (MAPK) activity and is part of the cAMP response element-binding protein (CREB) pathway that can lead to synaptic restructuring to support LTP. Here, we explored whether persistent activation of Ras in neurons further enhances LTP following EE of rodents. Immediately following weaning, transgenic mice that expressed constitutively activated neuronal Ras, or their wildtype (Wt) littermates, underwent 3weeks of constant EE. In the absence of EE, theta burst stimulation (TBS) evoked LTP in the CA1 region of transgenic mice that was not significantly different from LTP in Wts. After 3weeks of EE, hippocampal LTP was improved in Wt mice. Enriched transgenic mice showed an equivalent level of LTP to enriched Wts, but it was not significantly different from non-enriched synRas controls. Western blot analysis performed after a pull-down assay showed that non-enriched transgenic mice expressed higher Ras activity compared to non-enriched Wts. Following EE, Ras activity was reduced in transgenics to levels detected in Wts. These results show that constitutive activation of Ras does not mimic the effects of EE on LTP. In addition, EE results in an equivalent enhancement of LTP transgenics and Wts, coupled with a decrease in Ras activity to Wt levels. This suggests that permanent activation of Ras in neurons of synRas animals following EE results in an altered feedback regulation of endogenous Ras activity that is not a key factor in LTP enhancements. The maintenance of Ras within a physiological range may thus be required for the optimization of LTP in the hippocampus.

Hippocampal function is compromised in an animal model of multiple sclerosis.

Multiple sclerosis (MS) is a progressive inflammatory autoimmune disease that is characterized by demyelination and axonal damage in the nervous system. One obvious consequence is a cumulative loss of muscle control. However, cognitive dysfunction affects roughly half of MS sufferers, sometimes already early in the disease course. Although long-term (remote) memory is typically unaffected, the ability to form new declarative memories becomes compromised. A major structure for the encoding of new declarative memories is the hippocampus. Encoding is believed to be mediated by synaptic plasticity in the form of long-term potentiation (LTP) and long-term depression (LTD) of synaptic strength. Here, in an animal model of MS we explored whether disease symptoms are accompanied by a loss of functional neuronal integrity, synaptic plasticity, or hippocampus-dependent learning ability. In mice that developed MOG35-55-induced experimental autoimmune encephalomyelitis (EAE), passive properties of CA1 pyramidal neurons were unaffected, although the ability to fire action potentials became reduced in the late phase of EAE. LTP remained normal in the early phase of MOG35-55-induced EAE. However, in the late phase, LTP was impaired and LTP-related spatial memory was impaired. In contrast, LTD and hippocampus-dependent object recognition memory were unaffected. These data suggest that in an animal model of MS hippocampal function becomes compromised as the disease progresses.

Long-term depression: a cellular basis for learning?

Long-term depression (LTD) comprises a persistent activity-dependent reduction in synaptic efficacy which typically occurs following repeated low frequency afferent stimulation. Hippocampal LTD has been a subject of particular interest due to the established role of the hippocampus in certain forms of information storage and retrieval. Recently, it was reported that LTD in the CA1 region may be associated with novelty acquisition in rats. CA1 LTD expression may also be increased in stressful conditions. This suggests a more complex role for this form of plasticity than the oft-cited hypothesis that it simply serves to prevent synapse saturation, by means, for example, of enabling reversal of long-term potentiation (LTP). One possibility is that LTD may be directly involved in the creation of a memory trace. Alternatively, LTD may prime a synapse in readiness for the expression of LTP, thereby contributing indirectly to information storage. There is increasing evidence that LTD is not mechanistically the reverse of LTP. Although some common processes exist, molecular, biochemical, electrophysiological and pharmacological studies all point to several quite distinct induction and maintenance mechanisms for this form of synaptic plasticity. Taken together these findings suggest that hippocampal LTD must be considered in a new light. This review focuses on the interpretation of novel and established information with regard to LTD in the hippocampal CA1 region in terms of its possible role as a cellular basis for learning and memory.

Priming of group 2 metabotropic glutamate receptors facilitates induction of long-term depression in the dentate gyrus of freely moving rats.

Activation of group 1 metabotropic glutamate receptors (mGluRs) has been shown to facilitate the induction of tetanically evoked long-term potentiation in vivo, whereas group 2 mGluR antagonists inhibit long-term depression (LTD) in the CA1 region. LTD has not been successfully demonstrated to date in the dentate gyrus of freely moving rats. In this study, it was found that 1-Hz low-frequency stimulation (LFS) when applied via a stimulating electrode chronically implanted in the perforant path, evoked short-term depression (STD) of field excitatory post-synaptic potentials and population spikes measured from the dentate gyrus granule cell layer. Application of the group 2 mGluR agonists (S)-4-carboxyphenylglycine (4C3HPG) or (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) prior to LFS facilitated the expression of LTD. Application of the group 2 mGluR antagonist (2S)-alpha-ethylglutamic acid (EGLU) prior to agonist application prevented the facilitating effect of the agonists on LFS-induced depression. EGLU did not influence the expression of LFS-induced STD. Neither 4C3HPG, DCG-IV nor EGLU had an effect on basal synaptic transmission at the concentrations used. The present study demonstrates that priming of group 2 mGluRs leads to induction of persistent LTD in the dentate gyrus in vivo, consistent with a role for group 2 mGluRs in metaplasticity.

Group III metabotropic glutamate receptors modulate long-term depression in the hippocampal CA1 region of two rat strains in vivo.

Hippocampal long-term depression (LTD) involves a long-lasting decrease in synaptic transmission which is induced by low-frequency stimulation (LFS). Evidence exists that variability in the responsiveness of rat strains to LFS occurs. Thus, Wistar rats readily express LTD in vivo, whereas Hooded Lister rats demonstrate at best short-term depression (STD) in response to LFS. Group III metabotropic glutamate receptor (mGluR)-involvement in the induction of LTD in freely moving rats has not yet been investigated. This study therefore examined the effect of group III mGluR activation and inhibition on LTD expression, and evaluated these effects in Wistar and Hooded Lister rats. Animals were chronically implanted with recording and bipolar stimulating electrodes in the CA1 region, and an injection cannula in the lateral cerebral ventricle. LFS (1 Hz, 900 pulses) induced LTD in Wistar, and STD in Hooded Lister rats. Agonist priming with L-2-amino-4-phosphonobutanoic acid (AP4, 400 nmol/5 microl) facilitated LTD expression in Hooded Lister but not Wistar rats. The antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine inhibited the facilitatory effects of AP4 in Hooded Lister- and impaired LTD expression in Wistar rats. These data imply a role for group III mGluRs in hippocampal LTD in vivo, and suggest that differences in this mGluR system may account, in part, for strain-dependent variations in LTD expression.

Group 1 metabotropic glutamate receptors contribute to slow-onset potentiation in the rat CA1 region in vivo.

It has been demonstrated in the CA1 region of the hippocampus in vitro, and in the dentate gyrus and CA1 region in vivo, that application of the metabotropic glutamate receptor (mGluR) agonist, 1S, 3R-amino cyclopentane 2,3-dicarboxylic acid triggers a slow-onset potentiation of synaptic transmission in the hippocampus. This study examined the involvement of group 1 and 2 mGluRs in this phenomenon in the CA1 region of freely moving rats. Drugs were applied via the lateral cerebral ventricle, and measurements were obtained from the CA1 region via permanently implanted electrodes. The group 1 mGluR agonists, 3,5-dihydroxyphenylglycine (DHPG, 20-100 nmol/5 microl) and trans-azetidine-2,4-dicarboxylic acid (ADA, 100 nmol-1 micromol/5 microl) induced a dose-dependent potentiation of basal synaptic transmission. The mGluR antagonist R,S-alpha-methyl-carboxyphenylglycine (MCPG, 1 micromol), and the group 1 mGluR antagonist, S-4-carboxyphenylglycine (4CPG, 100 nmol) completely inhibited the effects of both DHPG and ADA. The group 2 mGluR agonist, (S)-4-carboxy-3-hydroxy phenylglycine (4C3H-PG, 50-200 nmol/5 microl) induced a dose-dependent decrease of basal synaptic transmission. These results suggest that in the CA1 region in vivo, slow-onset potentiation may be mediated by group 1 mGluRs.

1S,3R-ACPD dose-dependently induces a slow-onset potentiation in the rat hippocampal CA1 region in vivo.

It has been reported that application of 1S,3R-1-aminocyclopentane 1,3-dicarboxylic acid (ACPD) in vitro triggers a slow-onset potentiation in the hippocampal CA1 region. This study examined the effect of ACPD in the CA1 of freely moving rats. No effect on fEPSP baseline recordings occurred at 40 and 400 microM/5 microliters, however at 4 mM/5 microliters ACPD induced a slow-onset potentiation. MCPG (200 mM/5 microliters) completely inhibited this ACPD effect. These results indicate that slow-onset potentiation in the CA1 region, also occurs in vivo.

Regional and developmental profile of modulation of hippocampal synaptic transmission and LTP by AP4-sensitive mGluRs in vivo.

L-AP4 is an agonist at the presynaptic metabotropic receptor subtypes mGluR4, mGluR6 and mGluR7. In vitro, L-AP4 has been shown to reduce glutamate release and thereby suppress hippocampal excitatory transmission. Little data is available with regard to the actions of this compound in vivo. This study examined the effects of L-AP4 injected i.c.v. in the hippocampus of freely-moving rats on synaptic transmission and long-term potentiation (LTP). Two age groups were employed: 8-week-old and 12-week-old. Administration of L-AP4, 80 mM/5 microliters, reduced evoked baseline responses in the dentate gyrus (DG) and CA1 of 8-week-old rats when compared with controls. This effect was blocked by MCPG. L-AP4, 40 mM/5 microliters, also reduced baseline in DG but not CA1. L-AP4 (20, 40 or 80 mM/5 microliters) had no effect on baseline in either DG or CA1 of 12-week-old animals. However, injection of L-AP4 (80 mM/5 microliters) significantly reduced the amplitude of LTP induced by tetanization in CA1 and DG. This effect was blocked by MCPG (200 mM/5 microliters). LTP reduction, tested in 12-week-old animals, also occurred with an L-AP4 concentration of 40 mM/5 microliters in CA1 but not in DG. These data indicate that L-AP4 inhibits LTP in vivo with a variation in sensitivity to the drug occurring between regions. It is suggested that the response in CA1 is produced by mGluR7, and in DG by presynaptic mGluR4 present on perforant path neurons. These results offer in vivo physiological evidence for a variation in functional response and in developmental regulation of these subtypes, dependent on the region of the hippocampus where they are located.

Group I mGlu receptors regulate the expression of the neuronal calcium sensor protein VILIP-1 in vitro and in vivo: implications for mGlu receptor-dependent hippocampal plasticity?

Metabotropic glutamate (mGlu) receptors are involved in several forms of synaptic plasticity in the rat hippocampus. Agonists which activate group I mGlu receptors induce slow-onset potentiation without prior tetanization in the hippocampal area CA1. Activation of group I mGlu receptors induces protein synthesis which may contribute to mGlu receptor-dependent forms of long-term plasticity. Calcium-binding proteins are widely considered to comprise key elements for synaptic plasticity. Therefore, we investigated whether the calcium sensor protein VILIP-1 is associated with group I mGlu receptor-mediated plasticity in the dentate gyrus (DG) in vivo.Application of either the group I and II mGlu agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD) or the selective group I agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) resulted in slow-onset potentiation in the DG of adult rats. In hippocampal cell cultures both agonists elicited an enhanced expression of VILIP-1. In situ hybridization revealed strong hippocampal expression of VILIP-1 and intracerebral application of DHPG to adult rats significantly enhanced hippocampal VILIP-1 expression. The DHPG effects in both, hippocampal cultures and in vivo, were prevented by the group I mGlu receptor antagonist 4-Carboxyphenylglycine (4CPG). Calcium sensor proteins thus appear to be regulated by mGlu receptors in an activity-dependent manner. A specific role for group I mGlu receptors is evident. Furthermore, the sensor proteins may function as molecular switches for the long-term regulation of synaptic plasticity.

Role of the group III metabotropic glutamate receptor in LTP, depotentiation and LTD in dentate gyrus of freely moving rats.

We investigated whether group III metabotropic glutamate (mGlu) receptors are critically involved in the expression of long-term potentiation (LTP), depotentiation, or long-term depression (LTD) in the dentate gyrus of freely moving rats. Male Wistar rats (7 8 weeks) underwent implantation of stimulating and recording electrodes in the medial perforant path and dentate gyrus granule cell layer, respectively. A cannula was permanently implanted into the ipsilateral cerebral ventricle to enable drug administration. Intracerebral injection of the group III mGlu receptor agonist, L(+)-2-amino-4-phosphonobutanoic acid (AP4), significantly inhibited LTP at a concentration which unaffects basal synaptic transmission. Depotentiation. short-term depression (STD) and LTDwere unaffected by the agonist. The antagonist. (R.S)-r-cyclopropyl-4-phosphonophenylglycine (CPPG), inhibited agonist effects. but had no independent effects on basal synaptic transmission. CPPG did not affect the profile of LTP, depotentiation or STD elicited by low frequency stimulation (LFS) at 0.5 or 3 Hz. but significantly impaired LTD expression (at I Hz) and STD elicited at 5 Hz. These findings suggest that activation of group III mGlu receptors is critically required for LTD. but not LTP or depotentiation in the dentate gyrus and provide evidence for the involvement of separate mechanisms underlying LTD and depotentiation.

ACPD-mediated slow-onset potentiation is associated with cell death in the rat CA1 region in vivo.

Slow-onset potentiation of synaptic transmission in the hippocampus in vitro and in vivo is induced by application of the metabotropic glutamate receptor (mGluR) agonist, 1S,3R-amino cyclopentane 2,3-dicarboxylic acid (ACPD). This study investigated the cellular response in the CA1 region of freely moving rats to ACPD application. Drugs were applied via the lateral cerebral ventricle, and measurements were obtained from the CA1 region via permanently implanted electrodes. ACPD (20 nmol/5 microl) produced a dose-dependent slow-onset potentiation in the CA1 region which lasted over 4 h. Histological evaluation at either 4 h or 7 days following ACPD-injection indicated that slow-onset potentiation was associated with gradual but marked cell death in the CA1 region. Whereas 20 nmol ACPD produced significant CAI neurotoxicity, concentrations which did not induce potentiation had little or no neurotoxic effect. Both the general mGluR antagonist R,S-alpha-methyl-carboxyphenylglycine (1 micromol/5 microl), and the group 1 mGluR antagonist (S)-4-carboxyphenylglycine (4CPG, 100 nmol/5 microl) significantly inhibited ACPD-induced neuropathology. In addition, 4CPG inhibited the expression of ACPD-mediated slow-onset potentiation. These results confirm previous findings that in the CA1 region in vivo, slow-onset potentiation is mediated group 1 mGluRs, and indicate that slow-onset potentiation may involve pathological processes.

Metabotropic glutamate receptor agonist trans-azetidine-2,4-dicarboxylic acid facilitates maintenance of LTP in the dentate gyrus in vivo.

We examined the role of metabotropic glutamate receptors by studying the effect of intracerebroventricular infusion of the putative mGluR agonist trans-azetidine-2,4-dicarboxylic acid (tADA) on long-term potentiation (LTP) in the dentate gyrus of freely moving rats. Weak tetanization caused a decremental potentiation which returned to baseline levels within 2 hr. Injections of tADA (20 mM/5 microliters) 30 min prior to weak tetanization prolonged LTP of the field EPSP for at least 24 hr.

An increased expression of the mGlu5 receptor protein following LTP induction at the perforant path-dentate gyrus synapse in freely moving rats.

The involvement of metabotropic glutamate (mGlu) receptors in the induction of long-term potentiation (LTP) in vivo has been consistently documented. We have investigated whether LTP induction in the dentate gyrus of rats leads to changes in expression of mGlu2/3 or -5 receptor subtypes in the hippocampus. LTP was induced at the medial perforant path-dentate gyrus synapses, and mGlu receptor expression was examined by Western blot or in situ hybridization. An up-regulation of mGlu5 receptors was observed in the hippocampus both 24 and 48 h following LTP induction. This effect was restricted to the dentate gyrus and CA1 region, whereas no changes in mGlu5 receptor protein (but an increase in mRNA levels) were observed in the CA3 region. The increased expression of mGlu5 receptors was directly related to the induction of LTP, because it was not observed when tetanic stimulation was carried out in animals treated with the NMDA receptor antagonist, 2-amino-5-phosphonopentanoate (AP5). Western blot analysis also showed a reduced expression of mGlu2/3 receptors in the whole hippocampus 24 h after LTP induction, indicating that the increased expression of mGlu5 receptors was specific. These data suggest that an up-regulation of mGlu5 receptors is a component of the plastic changes that follow the induction of LTP at the perforant path-dentate gyrus synapse.

Metabotropic glutamate receptor subtype agonists facilitate long-term potentiation within a distinct time window in the dentate gyrus in vivo.

Trans-azetidine-2,4-dicarboxylic acid (ADA) is a putative selective agonist of group 1 metabotropic glutamate receptors. It has been shown previously that application of ADA prior to a short-term potentiation-inducing high-frequency tetanus facilitates long-term potentiation in vivo. In order to examine the role of metabotropic glutamate receptors in this response, we studied the effect of ADA in the dentate gyrus of the rat when applied after high-frequency tetanus to the perforant path. A comparison was made with the effects of the metabotropic glutamate receptor group 1 agonist 3,5-dihydroxyphenylglycine. Drugs were applied via a cannula implanted in the lateral cerebral ventricle. Both population spike amplitude and field excitatory postsynaptic potential were measured. Weak tetanization produced a short-term potentiation of field excitatory postsynaptic potential and population spike which decayed to baseline values by 90 min, and was unaffected by vehicle injections. Application of ADA (20 mM/5 microliters) or 3,5-dihydroxyphenylglycine (4mM/5 microliters) 5 min after high-frequency tetanus facilitated short-term potentiation into a long-term potentiation which lasted over 24 h. (R,S)-alpha-Methyl-4-carboxyphenylglycine (200 mM/5 microliters), a metabotropic glutamate receptor antagonist, when applied prior to high-frequency tetanus and ADA or 3,5-dihydroxyphenylglycine, completely inhibited this effect. ADA applied 10,15,20 and 25 min after high-frequency tetanus also facilitated short-term potentiation into long-term potentiation, but the magnitude of long-term potentiation was smaller than than produced by ADA given 5 min after tetanus. Similar effects were seen with 3,5-dihydroxyphenylglycine applied 25 min after high-frequency tetanus. When (R,S)-alpha-methyl-4-carboxyphenylglycine was applied prior to high-frequency tetanus and ADA or 3,5-dihydroxyphenylglycine applied 30 min after high-frequency tetanus, or after short-term potentiation decay, elicited no facilitation of long-term potentiation. These results indicate that a distinct time window for the enhancement by ADA and 3,5-dihydroxyphenylglycine of short-term potentiation into long-term potentiation occurs in the dentate gyrus in vivo. This suggests that metabotropic glutamate receptor activation in long-term potentiation occurs within a finite period of time and may be mediated by group 1 metabotropic glutamate receptors. Furthermore, it suggests that metabotropic glutamate receptor modulation of N-methyl-D-aspartate receptors does not account for the role of metabotropic glutamate receptors in long-term potentiation.

In vivo electrophysiological investigations into the role of histamine in the dentate gyrus of the rat.

Drugs acting at the three known classes of histamine receptors were injected intracerebroventricularly into the rat. The effects of these drugs upon synaptic potentials recorded from the dentate gyrus of the freely-moving rat were determined. Population spikes and field excitatory postsynaptic potentials were recorded from the granule cell layer of the dentate gyrus following stimulation of the perforant path. Drugs, dissolved in 0.9% NaCl were applied into the lateral cerebral ventricle in a volume of 5 microl over a period of 6 min. The histamine H1 receptor antagonist mepyramine (0.4 or 0.8 microg) had no significant effect on population spikes or field excitatory postsynaptic potentials. In contrast the H2 receptor antagonist cimetidine (3.25, 6.5 or 13 microg) showed a biphasic effect. At the lower doses (3.25 or 6.5 microg) a small (15%) depression of the field excitatory postsynaptic potentials and population spikes was observed beginning about 1 h following the infusion. At the highest dose tested (13 microg) a marked increase of the population spike was observed beginning immediately following the infusion and lasting for 90 min. Application of the H3 receptor agonist R-alpha-methylhistamine (0.2 microg) depressed the field excitatory postsynaptic potentials (15% at 4 h post-injection) and even more strongly the population spike (50%). Surprisingly, at higher doses (0.4 and 0.8 microg) no effect was seen. The H3 receptor antagonist thioperamide (0.41 and 0.82 microg) did not cause an increase in synaptic potentials but rather at the highest dose a small depression occurred at later time points (2-4 h following the infusion). At the lower dose (0.41 microg) thioperamide blocked the effect of R-alpha-methylhistamine (0.2 microg). These results show that the histaminergic system modulates information flow through the dentate gyrus in a complex manner involving both histamine H2 and H1 receptors.