Comparison of Y-receptor subtype expression in the rat hippocampus

Regulatory Peptides 75–76 (1998) 109–115

Comparison of Y-receptor subtype expression in the rat hippocampus Rachel M.C. Parker, Herbert Herzog* Neurobiology Program, Garvan Institute of Medical Research, St Vincent’ s Hospital, 384 Victoria Street, Darlinghurst, NSW 2010, Australia Received 12 October 1997; received in revised form 2 March 1998; accepted 3 March 1998

Abstract The mRNA expression patterns for the Y 1 , Y 2 , Y 4 and Y 5 receptor subtypes have been compared at a cellular level within consecutive coronal sections of rat hippocampus, using a uniform method of in situ hybridisation. All four receptor subtypes show different levels and patterns of expression. The Y 5 receptor mRNA is most abundant and most widely distributed (CA3 . DG ¯ CA2 ¯ CA1). Numerous Y 5 mRNA-expressing neurons are also observed in the dentate gyrus polymorphic layer, while several positively-labelled neurons are detected in the molecular layer and in the stratum oriens. The Y 2 receptor displays lower hybridisation signal relative to the Y 5 , although the expression pattern is similar. Moderate levels of Y 1 mRNA expression are detected in the pyramidal cell layer of CA3–CA1 fields. However, only 20% of dentate gyrus granule neurons express Y 1 receptor mRNA. In contrast, Y 4 receptor mRNA is much less abundant, only small subpopulations of Y 4 receptor expressing neurons are detected within the dentate gyrus and the CA3 to CA1 fields. This differential Y-receptor subtype expression pattern indicates specific and separate roles for these receptors in hippocampus processing, which may prove important in dysfunctional states, such as epilepsy and Alzheimer’s disease.  1998 Elsevier Science B.V. All rights reserved. Keywords: Y 1 , Y 2 , Y 4 and Y 5 receptor; In situ hybridisation; Expression; Epilepsy; Alzheimer’s disease

1. Introduction The hippocampus is an important centre for regulation and processing of memory, learning and emotional behaviour [1]. These functions are believed to be elicited through it’s association with the amygdala, entorhinal cortex and the hypothalamo–hypophysial axis. Hippocampal damage parallels a wide range of clinical disorders, including short-term and spatial memory loss, Alzheimer’s Dementia and temporal lobe epilepsy, for which the precise mechanisms are still unknown. Neuropeptide Y-immunoreactivity (NPY-ir) within the rat and human hippocampus has been localised to numerous non-pyramidal cell bodies (particularly within the polymorphic layer of the dentate gyrus; DG) and to extensive networks of axons and terminals innervating a variety of neuronal populations [2]. NPY causes presynap*Corresponding author. Tel.: 1 61 2 92958296; fax: 92958281; e-mail: [email protected]

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tic inhibition of excitatory neurotransmission in normal rat hippocampal slices, and in slices which show uncontrolled neuronal excitation representing models of limbic epileptic seizures [3,4]. Furthermore, NPY knockout mice are more susceptible to spontaneous seizures than wild-type [5] and long-lasting upregulation of NPY mRNA and NPY-ir are seen within the hippocampus of rat in vivo models of limbic epilepsy [6]. These findings are consistent with a role for NPY in controlling hippocampal excitability. NPY-mediated excitation of rat hippocampal neurons has also been reported [7,8], suggesting NPY may have several different mechanisms of action within this region. In addition, levels of hippocampal NPY are disturbed in other clinical disorders associated with the hippocampus, such as Alzheimer’s and Senile Dementia [9]. NPY mediates its effects through a large family of related G-protein-coupled receptors [10]. There are six Y-receptors known to date, five of which have been cloned (Y 1 , Y 2 , Y 4 , Y 5 and y6; with the y6 receptor being a truncated nonfunctional form in primates). Autoradiog-

0167-0115 / 98 / $ – see front matter  1998 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 98 )00059-7

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raphic studies, using [ 125 I]-NPY or [ 125 I]-PYY (high affinity ligands for most of the cloned Y-receptor subtypes) reveal an abundant, yet heterogeneous, population of Yreceptor binding sites within the hippocampus, particularly concentrated within the stratum oriens and stratum radiatum of the CA fields [11,12]. Unfortunately, the lack of selective ligands (agonists and antagonists) as well as subtype-specific antibodies for the different Y-receptor subtypes makes it very difficult to investigate the proportion and importance of specific Y-receptor subtypes in hippocampal processing. In this study we have used in situ hybridisation to compare the mRNA expression pattern of the Y 1 , Y 2 , Y 4 and Y 5 receptor subtypes within consecutive coronal sections of rat hippocampus, allowing cellular localisation of site of synthesis of each of these subtypes, whilst circumventing the ambiguity created in ligand binding studies due to the lack of selective pharmacological tools.

pcDNA3 vector (Invitrogen Corp., USA). Each cDNA sequence has 100% identity with the corresponding receptor gene sequence, but showed less than 60% homology with other known base sequences. For each construct, conditions for in vitro transcription were as follows; approximately 50 ng / ml linearised phenol / chloroformpurified template cDNA, 40 mM Tris–HCl (pH 8), 6 mM MgCl 2 , 10 mM dithioerythritol (DTE), 2 mM spermidine, 10 mM NaCl, 5 mM dithiothreitol (DTT), 1 unit / ml RNase inhibitor, 1 mM ATP, 1 mM CTP, 1 mM GTP, 0.65 mM UTP, 0.35 mM 11 digoxigenin-UTP (Boehringer Mannheim, Germany) and 2 units / ml of either T3, T7 or SP6 RNA polymerase (Boehringer Mannheim, Germany). After incubation at 378C for 2 h, the DNA template was digested with 1 unit / ml RNase-free DNase I and each riboprobe purified from unincorporated label, using a Sephadex G25 spun column. Yield was assessed on a dot blot.

2.3. In situ hybridisation histochemistry 2. Materials and methods

2.1. Tissue preparation Adult male Wistar rats (300–350 g; Animal Research Centre, Perth, Australia) were housed in a controlled environment under a regulated 12 h light–dark cycle and given food and water ad libitum. Four rats were anaesthetised with sodium pentobarbitone (i.p. at a dose of 60 mg / kg; Nembutal; Boehringer Mannheim, Germany), then killed by cardiac perfusion with ice cold 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS) (pH 7.4). Tissue was immediately dissected, postfixed in the same fixative, dehydrated in ethanol, paraffin wax embedded then stored at 48C until use. Rats were routinely killed between 10 am and 12 noon to minimise any possible effects due to diurnal changes in mRNA expression. Serial 5 mm coronal brain sections were cut on a microtome, slide-mounted and stored at 48C until use.

2.2. Riboprobe preparation Antisense and sense riboprobes were generated for the rat Y 1 , Y 4 and Y 5 receptor subtypes from selected cDNA regions corresponding to part of the coding sequence of each receptor (Y 1 antisense and sense corresponding to base sequence 59–654 [13]; Y 4 antisense and sense corresponding to base sequence 820–1146 [14]; Y 5 antisense corresponding to base sequence 461–1080 and sense corresponding to base sequence 1–461 [15]; where 1 represents the first base of the translation initiation codon), subcloned into pBluescript plasmid vectors (Stratagene, USA). Y 2 antisense and sense riboprobes were generated from cDNA corresponding to base sequence 838–1177 (antisense) and to base sequence 1–308 (sense) of the human Y 2 receptor sequence [16], subcloned into a

Precautions were taken throughout the procedure to avoid RNase contamination. Sections were brought to room temperature (RT), dewaxed in histoclear (2 3 4 min), rehydrated through decreasing concentrations of ethanol (from 100% to 50%), washed in 0.1 M PBS and incubated with 5 mg / ml of proteinase K (Boehringer Mannheim, Germany), 50 mM Tris–HCl (pH 7.5) and 5 mM EDTA, at 378C in a humid environment. Proteinase K treatment was stopped by incubation in 0.1 M PBS containing 0.1 M glycine. Sections were acetylated with 150 mM NaCl, 1.5% (vol / vol) triethanolamine, 0.25% (vol / vol) acetic anhydride to reduce non-specific background, then washed in 0.1 M PBS before in situ hybridisation. Sections were incubated with hybridisation buffer (pH 7–7.5), containing 50% formamide, 10% (wt / vol) dextran sulphate, 300 mM NaCl, 20 mM NaPO 4 , 2 mM EDTA, 1 3 Denhardt’s solution, 100 mg / ml yeast tRNA, 10 mM DTT and up to 600 ng / ml of digoxigenin-labelled riboprobe. Expression of the different subtype mRNAs was simultaneously assessed in serial sections within the same assay, for subsequent parallel comparisons of signal intensity. Hybridisation was carried out under coverslips overnight at 458C in a humid environment. Coverslips were then removed in 2 3 SSC at RT (1 3 SSC 5 0.15 M sodium chloride, 0.015 M trisodium citrate, pH 7.0) and sections washed in 0.2 3 SSC (558C for 30 min), 0.1 3 SSC (608C for 30 min), incubated with RNaseA (20 mg / ml in 0.5 M NaCl, 10 mM Tris–HCl, 1 mM EDTA at pH 8) at 378C for 15 min, washed in 2 3 SSC at RT for 5 min and finally in 0.2 3 SSC (378C for 30 min). Sections were incubated with 1.5 units / ml sheep anti-digoxigenin Fab antibody fragments conjugated with alkaline phosphatase, 2% skimmed milk powder, 150 mM NaCl, 100 mM Tris–HCl (pH 7.4), for 2 h in a humid environment. Alkaline phosphatase was then detected at RT, using 188 mg / ml 5-bromo-4chloro-3-indolyl phosphate (BCIP) and 375 mg nitroblue

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tetrazolium (NBT) substrate, containing 1 mM levamisole to inhibit endogenous alkaline phosphatase activity. The colour reaction was stopped in several washes of 10 mM Tris–HCl, 1 mM EDTA buffer (pH 8) and the sections immediately mounted under coverslips.

2.4. Analysis Positive hybridisation was detected and counted under light microscopy at 3 400 magnification using the following criteria: (i) colour precipitate showed a halo pattern in the cytoplasm surrounding a clearly visible nucleus and (ii) the pattern of positive hybridisation was only detected when the antisense riboprobe was employed and not with the corresponding sense strand. Regions of positive hybridisation were mapped with the aid of a rat brain atlas [17]. The number of mRNA-expressing neurons in sections from three rats were counted, using BioQuant Image Analysis software, across the entire field of view at 3 400 magnification and were represented as a percentage of the total population counted within that field. Nomarsky optics were employed to aid detection of non-positively labelled neurons. Adjacent sections were counterstained with Haematoxylin / Eosin to confirm morphological identification.

3. Results The expression of Y 1 , Y 2 , Y 4 and Y 5 receptor mRNA’s has been analysed and compared in consecutive coronal sections of the rat hippocampus, using highly subtype specific riboprobes and employing a uniform technique of in situ hybridisation histochemistry. None of the corresponding sense riboprobes used under identical assay conditions show specific hybridisation (Figs. 1 and 2). Furthermore, no cross-reactivity occurs when each of the riboprobes are hybridised against the other Y-receptor constructs under identical conditions to those for in situ hybridisation. This indicates probe selectivity at least for the cloned Y-receptor subtypes known to date, although cross-reactivity with as yet unidentified Y-receptors cannot be completely ruled out. The sensitivity of the assay was also assessed in different cell lines stably transfected with each Y-receptor subtype. Similar concentrations of these riboprobes show equivalent levels of signal in the corresponding cell lines when identical conditions to those for tissue are employed, indicating the comparability of this method. No signal is detected in these cell lines when the sense strand is used, nor when non-transfected cells are examined (data not shown). The Y 5 receptor shows the highest level of mRNA expression compared to the Y 1 , Y 2 and Y 4 receptor subtypes, not only in terms of amount of hybridisation signal (Figs. 1 and 2) but also in numbers of expressing neurons (Table 1). Over 80% of neurons throughout the

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pyramidal layer of CA3 express a very high level of Y 5 receptor mRNA. A lower, yet substantial, level of expression can be detected in 50% to 60% of neurons in the pyramidal cell layers of CA2 and CA1 and also in the granule cell layer of the dentate gyrus. Numerous Y 5 receptor mRNA-expressing neurons are also detected in the polymorphic layer of the dentate gyrus (Fig. 1), whilst positively-labelled cells are only very rarely detected within the molecular layer of this region. Occasional Y 5 receptor mRNA-expressing neurons can be seen in the stratum oriens cell layer, adjacent to the pyramidal cell layer. The distribution and number of Y 2 receptor mRNA expressing neurons is very similar to that obtained for Y 5 receptors, although the overall Y 2 receptor mRNA expression level is clearly lower (Fig. 1, Fig. 2 and Table 1). Y 2 receptor mRNA expression within pyramidal neurons is highest in the CA3 (over 70% of neurons express Y 2 mRNA), with similar levels being found in CA2, CA1 and the granule cell layer of the DG. Interestingly, the number of positive neurons in the pyramidal layers of the CA2 and CA1 fields is comparable to those found for the Y 5 receptor subtype. Whereas, compared to Y 5 , there are fewer, less intensely-labelled Y 2 receptor mRNA-expressing neurons in the granule cell layer of the DG (Fig. 1 and Table 1). Positively-labelled cells for the Y 2 receptor mRNA are also observed in the striatum oriens of the CA fields, as well as in the polymorphic layer, especially in the upper blade of the dentate gyrus, with only one or two Y 2 receptor mRNA-containing cells being observed within the molecular layer. However, Y 2 receptor mRNA expressing cells are found in the stratum radiatum between CA2 and CA3 in more caudal areas of the hippocampus. In contrast to the Y 2 and Y 5 receptors, the Y 1 receptor subtype shows a much more uniform and substantially lower level of mRNA expression throughout the pyramidal cell layer of the CA regions, in both signal intensity and cell numbers (Fig. 1, Fig. 2 and Table 1). On average, 40% to 55% of pyramidal neurons express detectable levels of Y 1 receptor mRNA, whereas Y 1 receptor mRNA expression is detected in only one fifth of granule layer cells of the dentate gyrus. Several Y 1 mRNA-expressing neurons are labelled in the polymorphic layer of the dentate gyrus, but these are negligible in the molecular layer of the dentate gyrus, or the stratum oriens and stratum radiatum layers of the hippocampus CA fields. Very low amounts of Y 4 receptor mRNA can be seen in small subpopulations of neurons throughout each subdivision of the hippocampus (Fig. 1, Fig. 2 and Table 1). The pyramidal cell layer of CA3 shows the highest number of detectable Y 4 receptor mRNA-expressing neurons. Here a third of all neurons display positive hybridisation as compared to the CA1 and CA2 regions, which express Y 4 receptor mRNA in only around 10% of neurons. On average 15% of granule cells of the dentate gyrus express Y 4 receptor mRNA. Y 4 receptor mRNA expression is not

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Fig. 1. Representative light field photographs of Y-receptor subtype mRNA expression in serial coronal sections (shown here; Y 1 vs Y 2 and Y 4 vs Y 5 receptor mRNA expression) of rat hippocampus at low power (scale bar 100 mm), as identified by in situ hybridisation histochemistry using the respective antisense riboprobes. Each of the sense probes shows no specific hybridisation, as represented here for the Y 1 receptor sense riboprobe. Location of the photographed area is highlighted in the schematic diagram (taken from [17]).

detectable in any other cell layers of the hippocampus, including the polymorphic and molecular layers of the dentate gyrus and the stratum oriens and radiatum of the CA fields. No variation can be seen in caudal as compared to rostral aspects of the hippocampus for each receptor subtype. An exception is the Y 1 receptor, where an apparent decrease in signal strength in the CA1 field can be seen at more caudal levels.

4. Discussion This is the first study which compares the mRNA expression of all four cloned functional Y-receptor subtypes in consecutive sections of the rat hippocampus on a cellular level using a uniform in situ hybridisation technique. Our results indicate a differential expression pattern of Y 1 , Y 2 , Y 4 and Y 5 receptor mRNA’s in rat hippocampus. These findings are in good agreement with previous

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Fig. 2. Representative light field photographs of neuronal Y-receptor subtype mRNA expression (filled arrows) in the CA3 (Y 1 , Y 2 and Y 5 receptor mRNA expression) and CA3–CA2 (Y 4 receptor mRNA expression) pyramidal cell layer of serial coronal sections (shown here; Y 1 vs Y 4 and Y 2 vs Y 5 receptor mRNA expression) of rat hippocampus at high power (scale bars 100 mm), as identified by in situ hybridisation histochemistry using the respective antisense riboprobes. None of the sense probes show positive hybridisation in any of the cell layers of the hippocampus, as represented here for the Y 4 receptor sense riboprobe; mol 5 molecular cell layer; gr 5 granule cell layer; CA3 and pm 5 CA3 field and polymorphic layer in the hilar area, respectively. Open arrows show nuclei of non-hybridising neurons. Location of the photographed area is highlighted in the schematic diagram (taken from [17]).

reports of cellular localisation for Y 2 mRNA [18] and Y 1 mRNA [19] except that the latter study describes high levels of Y 1 mRNA in the dentate gyrus granule cell layer, an area where we find relatively lower levels of Y 1 mRNA and which displays relatively few Y-receptor binding sites

[11,12], or BIBP3226-sensitive binding sites (highly selective Y 1 receptor antagonist) [20]. This variation may be due to differences in sensitivity of the in situ hybridisation techniques employed in that as compared to our study. High levels of Y 5 mRNA in rat hippocampus [21], and low

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Table 1 Mean percentage of positively-labelled neurons for each of the four Y-receptor subtypes out of the total number of neurons in areas of the hippocampus identified using defined criteria [17,23] Region

Dentate gyrus granule cell layer CA3 pyramidal cell layer CA2 pyramidal cell layer CA1 pyramidal cell layer

Percentage of Y-receptor subtype mRNAexpressing neurons Y1

Y2

Y4

Y5

22.661.6

38.062.1

14.961.4

50.662.0

54.262.2

70.962.9

31.464.3

82.764.9

39.065.7

45.162.6

6.261.7

60.265.7

37.163.4

54.363.1

10.361.7

49.267.2

Serial sections were analysed for expression of each subtype, focusing on the rostrocaudal positions from Bregma 2 3.80 mm to 2 5.80 mm. Values represent %6S.E. for n 5 3 rats.

levels of Y 4 mRNA in the human hippocampus [22] have also previously been reported, although not at a cellular level. Our results indicate the majority of CA field pyramidal neurons express Y 2 and / or Y 5 mRNA, with the CA3 pyramidal neurons expressing the highest proportion of each of the four receptor mRNAs. This abundance of Y-receptor mRNA expression is in accordance with the high densities of Y-receptor binding sites observed in the hippocampus [11,12]. Moreover, these binding sites are particularly concentrated in the stratum oriens and stratum radiatum layers of the CA fields; layers containing numerous synapses between dendritic trees of the pyramidal layer neurons and terminals from other pyramidal and granule neuron projections [23] and importantly also contain many synaptic contacts with NPY-ir terminals [2,24]. In addition, electrophysiology experiments show the majority, if not all, of the excitatory hippocampal neurons recorded from culture and slice preparations respond to NPY [8,25,26]. Studies using various competing ligands have stated that half the population of [ 125 I]PYY binding sites can be blocked by [Leu 31 , Pro 34 ]NPY (Y 1 / Y 4 / Y 5 agonist) and half can be blocked by NPY 13 – 36 (a Y 2 agonist) [19]. This suggests that Y 2 receptor subtypes are the predominant known Y-receptor subtype in the rat hippocampus, a proportion which is not reflected in the comparative mRNA expression levels found in our study. It may be that other unknown Y subtypes make up a fraction of this binding, or that the level of mRNA expression is not a direct correlate of receptor protein levels in these areas. It is conceivable that a fraction of the detected Y-receptor mRNA for any of the four subtypes is actually expressed as receptor protein on distant axons and terminals, which are known to project from pyramidal neurons to other intrahippocampal and extrinsic regions, including the lateral septal nucleus [23].

Interestingly, one study finds negligible amounts of [ 125 I]hPP (Y 4 / Y 5 agonist) binding sites in the rat hippocampus [27], which is surprisingly in view of the high levels of Y 5 receptor mRNA expressed here. As yet, no selective tools are available to definitively study the cellular localisation of each of the Y-receptor subtypes. Electrophysiological studies also provide substantial evidence that Y-receptor subtypes with pharmacological profiles indicative of Y 2 and Y 5 receptors play a major role in presynaptic inhibition of excitatory neurotransmission to the pyramidal neurons in normal and in epileptic seizure states [3,4,26,28]. The distribution of Y 2 and Y 5 receptor mRNA we report is consistent with autoradiography and electrophysiology results, which show Y 2 and Y 5 receptors are located on the excitatory terminals of Schaffer collaterals of pyramidal neurons and to a lesser extent of mossy fibres of granule cells, where they act to control the feed-forward excitation of these neurons. Surprisingly, mRNA for the PP-preferring Y 4 receptor subtype [14] can also be found in several discrete areas of the rat hippocampus at low levels. The pharmacological profile of NPY’s anticonvulsive actions within the hippocampus does not rule out an involvement of the Y 4 subtype [28,30]. The functional role of Y 1 mRNA, which we clearly demonstrate to be expressed in discrete hippocampal neuron populations, still remains to be determined. It has been suggested that Y 1 receptors may have a pronounced role in hippocampal development and early maturation [29]. Putative Y 1 receptor binding sites are affected in kainate treated rats [31,32], despite the reported lack of Y 1 receptor-mediated anticonvulsive activity [28]. Interestingly, excitatory responses to NPY application have been noted in approximately 6% of hippocampal neurons recorded in culture [8]. NPY application also directly elicits excitatory responses in rat granule cells in vitro [7], dendrites of which appear to be in direct synaptic contact with NPY-ir terminals [33]. However, the pharmacological profiles of these excitatory responses have not been studied and therefore the possible involvement of Y 1 and Y 4 receptor subtypes in these effects remains unknown. The hippocampus contains numerous subpopulations of neurons and a high complexity of connectivity [23]. It will be important to phenotypically-characterise Y-receptor-expressing neurons in these regions in more detail in order to further elucidate their functions. Furthermore, the abundance of each Y-receptor subtype mRNA in certain regions, notably in CA3 pyramidal neurons, suggests there may be some overlap in expression within the same neurons. Consequently, co-expression studies may reveal distinct neuronal populations. Hippocampal Y-receptor binding sites are modulated in models of limbic seizure [31,32]. Therefore, it may be appropriate to investigate possible modulation of specific Y-receptor subtype mRNA levels in these models, to determine the possible importance of one subtype over others in such physiologically relevant situations.

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In the light of a growing number of Y-receptor subtypes and the lack of subtype-selective pharmacological tools to study their mechanisms of action, our study provides important additional information on their significance within hippocampus processing.

[16]

[17]

Acknowledgements

[18]

This work was supported by Bristol–Myers–Squibb in collaboration with the Garvan Institute for Medical Research.

[19]

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