Orexin-saporin lesions of the medial septum impair spatial memory

Neuroscience 132 (2005) 261–271

OREXIN-SAPORIN LESIONS OF THE MEDIAL SEPTUM IMPAIR SPATIAL MEMORY H. R. SMITHb AND K. C. H. PANGa,c*

lesions had difficulty in learning the water maze (Morris et al., 1982) and the radial maze (Olton et al., 1979) tasks. Damage of the medial septum and diagonal band of Broca (MSDB), which connects to the hippocampus via the fimbria– fornix pathway (Jakab and Leranth, 1995), also impairs learning and memory. Transections of the fimbria–fornix pathway result in deficits in spatial learning and memory similar to those seen with direct hippocampal damage (Becker et al., 1980; McDonald and White, 1994; Everitt and Robbins, 1997; Cassel et al., 1998). Electrolytic lesions of the medial septal area impair spatial (Winson, 1978) and order memory tasks (Kesner et al., 1986). In addition, excitotoxic lesions of the MSDB impair place navigation in a water maze task (Hagan et al., 1988) and other spatial memory tasks (Hepler et al., 1985a, b). Finally, temporary inactivation of the medial septal region results in a drastic impairment of memory and loss of hippocampal ␪ rhythm, an electroencephalography (EEG) activity pattern postulated to be important for memory formation (Givens and Olton, 1990; Mizumori et al., 1990; Hasselmo et al., 2002). Therefore, the role of the MSDB and hippocampus in spatial learning and memory is well established. The MSDB projection to hippocampus provides the only known cholinergic input to the hippocampus (McKinney et al., 1983) and also includes a considerable non-cholinergic component (Wainer et al., 1985; Gaykema et al., 1990). The non-cholinergic component is mostly composed of GABAergic neurons that innervate hippocampal GABA interneurons (Freund and Antal, 1988; Kiss et al., 1990; Jakab and Leranth, 1995). These GABAergic septohippocampal neurons have been further characterized as containing the calcium binding protein parvalbumin (PV; Freund, 1989). The MSDB also contains a smaller number of peptidergic neurons, including those that contain substance-P, calcitonin-gene related peptide, enkephalins, and luteinizing hormone releasing hormone (Jakab and Leranth, 1995). Thus, the MSDB is a heterogeneous collection of neurons, with the majority of septohippocampal neurons consisting of cholinergic and GABAergic neurons. In assessing the importance of the MSDB in learning and memory, much of the focus has been on the cholinergic neurons. Development of the immunotoxin, 192-IgGsaporin, has facilitated research into the function of these cholinergic neurons (Wiley et al., 1991). 192-IgG-saporin consists of the antibody to the rat p75 nerve growth factor receptor conjugated to saporin, a ribosome-inactivating protein. Cholinergic neurons in the MSDB are the only neurons in this region to contain the p75 receptor, and therefore, injections of 192-IgG-saporin into the MSDB

a

J. P. Scott Center for Neuroscience, Mind and Behavior, Bowling Green State University, Bowling Green, OH 43403, USA b Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403, USA c

Department of Psychology, Bowling Green State University, Bowling Green, OH 43403, USA

Abstract—The medial septum and diagonal band of Broca (MSDB) provide a major input to the hippocampus and are important for spatial learning and memory. Although electrolytic MSDB lesions have prominent memory impairing effects, selective lesions of either cholinergic or GABAergic MSDB neurons do not or only mildly impair spatial memory. MSDB neurons are targets of orexin-containing neurons from the hypothalamus. At present, the functional significance of orexin afferents to MSDB is unclear, and the present study investigated a possible involvement of orexin innervation of the MSDB in spatial memory. Orexin-saporin, a toxin that damages neurons containing the hypocretin-2 receptor, was administered into the MSDB of rats. Rats were subsequently tested on a water maze to assess spatial reference memory and a plus maze to assess spatial working memory. At 100 ng/␮l, orexin-saporin destroyed primarily GABAergic septohippocampal neurons, sparing the majority of cholinergic neurons. At 200 ng/␮l, orexin-saporin almost totally eliminated GABAergic septohippocampal neurons and destroyed many cholinergic neurons. Spatial reference memory was impaired at both concentrations of orexin-saporin with a dramatic impairment observed for 24-h retention. Short-term reference memory was also impaired at both concentrations. Rats treated with 200 ng/␮l, but not 100 ng/␮l, of orexinsaporin were also impaired on a spontaneous alternation task, showing a deficit in spatial working memory. Our results, together with previous studies, suggest that orexin innervation of the MSDB may modulate spatial memory by acting on both GABAergic and cholinergic septohippocampal neurons. © 2005 Published by Elsevier Ltd on behalf of IBRO. Key words: acetylcholine, GABA, hippocampus, learning, hypocretin.

Previous research has identified the hippocampus and medial septum as important in spatial learning and memory. Damage to the hippocampus produces deficits of spatial learning and memory. For instance, rats with hippocampal *Correspondence to: K. Pang, Department of Psychology, Bowling Green State University, Bowling Green, OH 43403, USA. Tel: ⫹1-419372-6974; fax: ⫹1-419-372-6013. E-mail address: [email protected] (K. Pang). Abbreviations: ChAT, choline acetyltransferase; DAB, 3,3= diaminobenzidine; EEG, electroencephalography; ir, immunoreactive; MSDB, medial septum/diagonal band of Broca; PB, phosphate buffer; PV, parvalbumin. 0306-4522/05$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2004.12.037

261

262

H. R. Smith and K. C. H. Pang / Neuroscience 132 (2005) 261–271

cause complete and selective damage of the cholinergic neurons in this region. Adjacent non-cholinergic neurons are unaffected (Wiley et al., 1991; Heckers et al., 1994). Despite the loss of cholinergic MSDB neurons with 192-IgG-saporin treatment, rats show small or no deficits on spatial memory tasks (Berger-Sweeney et al., 1994; Baxter et al., 1995; Bannon et al., 1996; Dornan et al., 1996; McMahan et al., 1997; Chappell et al., 1998; Pang and Nocera, 1999). Although some studies have found impairments following intraseptal 192-IgG-saporin (Leanza et al., 1995; Walsh et al., 1996; Janis et al., 1998; Wrenn et al., 1999; Lamprea et al., 2000; Lehmann et al., 2002; Johnson et al., 2002; Chang and Gold, 2004), the loss of cholinergic neurons with 192-IgG-saporin is greater than that seen with non-selective excitotoxic lesions, yet the behavioral impairment is much less dramatic with 192-IgGsaporin than with excitotoxic lesions (Dunnett et al., 1991; Waite and Thal, 1996). Therefore, it has been suggested that damage to non-cholinergic neurons may be responsible for the profound impairments of learning and memory observed following excitotoxic lesions (Robbins et al., 1989; Markowska et al., 1990; Dunnett et al., 1991). Little research has investigated the role of noncholinergic MSDB neurons in spatial learning and memory. Kainic acid, in appropriate concentrations, can preferentially destroy GABAergic neurons with minimal damage to the cholinergic neurons in the septum (Malthe-Sorenssen et al., 1980; Walaas, 1981). Some loss of cholinergic neurons was seen when kainic acid was injected into the diagonal band of Broca (Malthe-Sorenssen et al., 1980); however, recent studies using lower concentrations of kainic acid showed a loss of PV-immunoreactive (ir) neurons (GABAergic septohippocampal neurons) with sparing of cholinergic neurons (Pang et al., 2001). Despite the almost complete loss of PV-ir neurons following the intraseptal injection of kainic acid, no behavioral impairment was seen on water maze or radial arm maze tasks (Pang et al., 2001). However, another study using intraseptal ibotenic acid reported impaired learning (Cahill and Baxter, 2001). In this study, ibotenic acid damaged PV-ir MSDB neurons while sparing cholinergic neurons. Rats treated with ibotenic acid were impaired in learning to enter a consistently baited arm of a T-maze from the same starting arm. Although damage of non-cholinergic MSDB neurons impaired learning of this task, the interpretation is complicated by the fact that hippocampal inactivation with lidocaine does not impair learning the same task, suggesting that ibotenic acid may have produced damage outside of the septohippocampal system (Packard and McGaugh, 1996). Thus, the limited studies currently available suggest that selective damage of septohippocampal GABAergic neurons do not impair spatial learning and memory. While lesions of either cholinergic or GABAergic septohippocampal neurons individually do not produce learning and memory deficits, nonselective lesions that damage both populations resulted in memory impairments. Electrolytic and ibotenic acid lesions that damage both neuronal populations impair spatial memory in the eight-arm radial maze (Kesner et al., 1986). Pang et al. (2001) used 192-

IgG-saporin and kainic acid to destroy both populations of MSDB neurons. Rats were trained on a water maze task, and those with lesions of both cholinergic and GABAergic septohippocampal neurons were slower to learn the location of the escape platform. Similar impairments were observed for working memory in a radial arm maze task (Pang et al., 2001). Recently, the hypocretin (also known as orexin) system has received much interest because of its importance in the sleep/wake cycle (Kilduff and Peyron, 2000). Hypocretin neurons are located in the lateral hypothalamus and have targets throughout the brain. One target is the medial septum where there is a dense collection of hypocretin-2 receptors (Nambu et al., 1999). The hypocretin-2 receptor is present on all PV-containing GABAergic neurons in the MSDB and possibly other neuronal populations, including the cholinergic septal neurons (Wu et al., 2002). Activation of the MSDB hypocretin receptors leads to excitation of both cholinergic and GABAergic septohippocampal neurons (Wu et al., 2002, 2004). To date, the functional role of the hypocretin/orexin innervation of the MSDB is unknown, although some have suggested a role in arousal (Wu et al., 2004). Damage to both GABAergic and cholinergic cells in the medial septum is produced by administration of the neurotoxin, hypocretin-saporin (Gerashchenko et al., 2001). Hypocretin-saporin (also known as orexin-saporin and referred to as orexin-saporin for the rest of the paper) is a toxin created by conjugating the hypocretin-2 receptorbinding ligand, hypocretin, to saporin. When injected into the MSDB, orexin-saporin produced extensive damage of both cholinergic and GABAergic cells, without affecting fibers of passage. Total elimination of hippocampal ␪ rhythm during locomotion resulted from orexin-saporin administration into the MSDB (Gerashchenko et al., 2001). Given the association of hippocampal ␪ rhythm and spatial memory and the importance of cholinergic and GABAergic septohippocampal neurons in learning and memory, we hypothesized that intraseptal orexin-saporin would impair spatial learning and memory. The primary aim of the present study was to determine whether the orexin innervation of MSDB has an important role in spatial memory. A secondary aim was to determine whether partial loss of both cholinergic and GABAergic septohippocampal neurons was sufficient to impair spatial memory. To accomplish these aims, three concentrations of orexin-saporin were infused into the MSDB of rats. Subsequently, rats were tested in the water maze and the spontaneous alternation tasks to assess spatial reference and working memory, respectively.

EXPERIMENTAL PROCEDURES Subjects Thirty-four male Long-Evans rats (250 –350 g; Harlan, Indianapolis, IN, USA) were used in this study. Animals were housed one or two to a cage in a colony room with a 12-h light/dark cycle. Testing was performed during the light phase. Procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved

H. R. Smith and K. C. H. Pang / Neuroscience 132 (2005) 261–271 by the Bowling Green State University Institutional Animal Care and Use Committee. All attempts were made to reduce the number of animals used and to minimize pain and suffering. Nine rats were treated with saline, and a total of 25 rats were treated with orexin-saporin. Eight rats received 100 ng/␮l, 11 rats received 200 ng/␮l, and six rats received 300 ng/␮l orexin-saporin. One saline rat could not complete behavioral testing due to motor difficulties in the water maze, and was not included in the data analysis. One 200 ng/␮l rat died during surgery, and a 100 ng/␮l rat died 2 days after surgery.

Surgery Rats were anesthetized with sodium secobarbital (50 mg/kg, i.p. supplemented as necessary throughout surgery). Scopolamine methyl bromide (0.02 mg/kg, i.p.; RBI, Natrick, MA, USA) was administered to reduce secretions. Body temperature was maintained at 37°C with a Deltaphase isothermal pad (BrainTree Scientific, Braintree, MA, USA). Each subject was placed in a stereotaxic instrument, and the head was leveled so that bregma and lambda were in the same horizontal plane. Holes (1–1.5 mm) were drilled in the skull over the MSDB. The tip of a Hamilton syringe was inserted in MS (0.6 mm anterior and ⫾0.5 mm lateral to bregma, 6.2 mm ventral to brain surface), and 0.3 ␮l of saline or orexin-saporin (100 –300 ng/␮l) was injected into each of the sites. Two additional injections, each of 0.4 ␮l, were administered into the left and right horizontal limbs of the diagonal band of Broca (0.6 mm anterior and ⫾0.5 mm lateral to bregma, 7.8 ventral to brain surface). The doses were administered at a rate of 0.05– 0.1 ␮l/min, and after each injection, the drugs were allowed to diffuse for 5 min before removal of the needle. The scalp was sutured and the animals were closely monitored for 2 days after the surgery. Subjects were allowed to recover for 12–14 days before behavioral testing began.

Behavior Water maze. Rats were trained in a water maze for 10 consecutive days. The water maze was a plastic circular container (1.5 m diameter) filled with water to a height of approximately 45 cm. The water was made opaque with nontoxic acrylic white paint. A clear Lucite platform (10 cm⫻10 cm) was placed 18 cm from the wall of the west quadrant (target quadrant). The level of the water was adjusted so that the platform was hidden 1.5 cm below the surface of the water. A variety of extramaze cues were located around the room, including posters, a sink, and doors. Each of the 10 daily sessions consisted of six trials. On each trial, the rat was placed in one of the non-target quadrants with its head facing the outer wall of the maze. Each of the non-target quadrants was used as the start location twice throughout a session. Each trial ended when the rat found the platform or was led to the platform by the experimenter after 60 s. After reaching the platform, the rat was left there for 15 s and then moved to a plastic holding container for 30 s. The time to reach the platform was recorded as the escape latency. On day 4 and day 11, rats were tested for their ability to remember the hidden platform location using a single probe trial on each day. The platform was removed from the maze, and rats were started in the quadrant opposite the target quadrant. The probe trial had a duration of 60 s and was recorded on videotape. Total time spent in the target quadrant, the latency time to the platform location, and the number of times the rat crossed the platform location were determined from offline analysis of the videotape. The probe trial on day 4 was completed 6 to 8 h before the training session on that day. Spontaneous alternation. The spontaneous alternation task was conducted in a room unfamiliar to the rats, using a wooden plus maze. Each of the four maze arms was 60 cm long and 10 cm

263

wide. The arms intersected in a 10 cm by 10 cm square. The maze was elevated 14 cm above the table to discourage rats from leaving the maze. Extramaze cues were located around the room, similar to ones described for the water maze. Rats were placed in the center of the maze, and allowed to freely explore the maze for one 20-min session. A video camera, located above the maze, recorded the rat’s movements. The experimenter, located in an adjacent room, observed the animal on a monitor. Arm choices were recorded as east, west, north and south. A choice was recorded when all four paws of the rat entered the arm.

Histology After behavioral testing was completed, each animal was perfused through the heart with 250 ml of 0.9% saline followed by 500 ml of chilled 4% paraformaldehyde in 0.1 M phosphate buffer (PB). After removal from the skull, the brain was post-fixed in 4% paraformaldehyde overnight at 4°C. The brain was then transferred to a 30% sucrose solution (pH 7.4) and left in this solution for 2 days. Using a freezing microtome, the brains were sectioned (50 ␮m) and then washed (3⫻5 min) in 0.1 M PB. Every third section was incubated in mouse anti-PV antiserum (1:1000; Sigma Immunochemicals, St. Louis, MO, USA), with 1.0% normal donkey serum, 0.5% Triton X-100, and 0.1% sodium azide in 0.1 M PB. Brain sections adjacent to those stained for PV were incubated in goat anti-choline acetyltransferase (ChAT) antiserum (1:500; Chemicon International, Temecula, CA, USA) with 1.0% normal donkey serum, 0.5% Triton X-100, and 0.1% sodium azide in 0.1 M PB. After incubation overnight at room temperature, the sections were washed in 0.1 M PB (3⫻5 min), and then incubated in the secondary antisera (Jackson Immunoresearch, West Grove, PA, USA), biotinylated donkey anti-mouse to bind to mouse anti-PV and biotinylated donkey anti-goat to bind to goat anti-ChAT. Secondary antisera consisted of 1:200 dilutions of antibody, 1.0% normal donkey serum, 0.5% Triton X-100, and 0.1% sodium azide in 0.1 M PB. After incubating for 2 h at room temperature, the sections were washed in 0.1 M PB (3⫻5 min), and then incubated in avidin/biotin solution for 2 h (1:200; standard ABC kit; Vector Laboratories, Burlingame, CA, USA). The tissue was then washed in 0.1 M PB (3⫻5 min) and incubated in nickel chloride (0.06%) enhanced 3,3= diaminobenzidine (DAB; 0.05%) for 20 min. Immediately following this incubation, hydrogen peroxide (final dilution of 0.0005%) was added to the nickel– DAB solution for 5–10 min to allow visualization of the cells. The tissue was washed a final time in 0.1 M PB (6⫻5 min). Brain sections were mounted on gelatin-coated microscope slides, dehydrated and cleared. Sections were covered with coverslip glass using Permount (Sigma). Drawings of the MSDB region with labeled neurons were constructed using the StereoInvestigator program (version 3.0; MicroBrightField, Colchester, VT, USA).

Data analysis All statistical analyses were performed with SPSS for Windows (version 11.0; SPSS, Inc., Chicago, IL, USA). Post hoc analyses were accomplished using Tukey’s HSD test, and significant interactions were evaluated using an F test. Mauchly’s tests of sphericity were conducted to determine violations in the assumptions of sphericity for repeated measures factors. The Greenhouse-Geisser correction was used in appropriate situations to correct for violations of sphericity (Geisser and Greenhouse, 1958).

Water maze The mean escape latency time was computed for each subject for each training day and analyzed using a mixed design ANOVA with

264

H. R. Smith and K. C. H. Pang / Neuroscience 132 (2005) 261–271

days as a within-subject factor and drug treatment as a betweensubjects factor. In order to assess long-term memory (i.e. 24-h memory), escape latency times for the first trial of each day were analyzed using a mixed design ANOVA with days as a withinsubject factor and drug treatment as a between-subjects factor. In order to assess short-term reference memory, the mean escape latency for each trial was calculated for the first day of training. The data were analyzed using a mixed design ANOVA with trials as a within-subject factor and drug treatment as a betweensubjects factor. Performance on the probe trials was analyzed using a mixed design ANOVA with the two probe sessions as a within-subject factor and drug treatment as a between-subjects factor. In addition, to determine the effects of orexin-saporin on performance early in the training process separately from performance later in training, the probe trials on days 4 and 11 were analyzed separately using a one-way ANOVA with treatment as the betweensubjects factor. Each of the three behavioral measures (time in target quadrant, the latency to the platform location, and the number of platform crossings) was analyzed separately.

Spontaneous alternation For each subject, five consecutive arm choices were used to determine whether an alternation occurred. An alternation was recorded if the rat entered all four possible arms in five consecutive choices. The number of actual alternations was divided by the total number of possible alternations (i.e. number of arms entered minus 4) in order to determine the alternation percentage for each animal. Animals with less than 10 possible alternations were excluded from the analysis. Percentages of alternation were analyzed using a one-way ANOVA with drug treatment as a between-subjects factor.

RESULTS Histology PV-ir and ChAT-ir neurons were found throughout the MSDB in rats injected with saline (Fig. 1, left). ChAT-ir neurons were located mostly lateral to the midline, whereas PV-ir neurons were primarily found along the midline of the MSDB region. These data are consistent with previous studies (Kiss et al., 1990; Pang et al., 2001). The effects of orexin-saporin were concentrationdependent. Rats receiving 100 ng/␮l orexin-saporin (orexin-100) had major damage to PV-ir neurons, but minor damage to cholinergic neurons (Fig. 1, center). Damage to PV-ir neurons was mostly in the MS with some sparing in the posterior horizontal limb of the diagonal band of Broca. Rats receiving 200 ng/␮l orexin-saporin (orexin-200) showed reduction of both PV-ir and cholinergic neurons (Fig. 1, right). These subjects had an almost complete elimination of the PV-ir neurons, and moderate loss of the ChAT-ir neurons. This is consistent with lesions reported in previous studies using 200 ng/␮l orexin-saporin (Gerashchenko et al., 2001). In both groups of rats (100 and 200 ng/␮l), cells in the lateral septum, overlying cortex and caudate-putamen appeared to be intact although a comprehensive examination was not conducted of areas outside the immediate vicinity of the MSDB. However, animals that received 300 ng/␮l orexin-saporin (orexin300) had extensive tissue damage in the medial septal area. The lateral ventricles were enlarged, and the target area had large holes or was completely missing from the

Fig. 1. Drawings of cholinergic (upper two rows) and PV-containing (bottom two rows) neurons in the MSDB region of control rats (left column), and rats treated with 100 ng/␮l (middle column) or 200 ng/␮l (right column) of orexin-saporin. An anterior and a posterior section of the MSDB are shown. For the control group, the distribution of neurons (closed squares) is drawn for a representative example. For rats treated with intraseptal injections of orexin-saporin, open circles represent the distribution of neurons from the rat with the least amount of damage, and the closed circles represent neurons from the rat with the most amount of damage in each treatment group.

brain slices. Because of the extensive damage to the tissue, behavioral data involving the orexin-300 treatment group are not reported. Water maze Both concentrations (100 and 200 ng/␮l) of orexin-saporin impaired the acquisition of the water maze task (Fig. 2). All rats showed some learning of the location of the platform as demonstrated by a significant main effect of days on escape latency, F(9,198)⫽82.562, P⬍0.001. Orexin-saporin significantly impaired learning as demonstrated by a main effect of treatment, F(2,22)⫽20.880, P⬍0.001, and a significant interaction of treatment and days, F(18,198)⫽ 1.901, P⫽0.018. However, the test for sphericity was violated, and following correction, the main effect of days continued to be significant (P⬍0.001) and the interaction approached significance (P⫽0.071). Post hoc analysis showed that both orexin-100 and orexin-200 differed from the saline group, but did not differ from one another.

H. R. Smith and K. C. H. Pang / Neuroscience 132 (2005) 261–271 60 55

Escape Latency (s)

50 45

Saline

40

Orexin 100

35

Orexin 200

30 25 20 15 10 5 0 1

2

3

4

5

6

7

8

9

10

Session Fig. 2. Acquisition of the water maze task following intraseptal administration of saline, 100 ng/␮l orexin-saporin or 200 ng/␮l orexinsaporin. Rats were trained with one session/day and performance was assessed using the time to reach a hidden platform (escape latency). Orexin-saporin (both concentrations) significantly impaired the acquisition of this task. No difference was found between the two groups receiving orexin-saporin.

To determine the effect of treatment on long-term reference memory, the mean escape latency times on the first trial of each training day were analyzed (Fig. 3). Performance on the first trials improved with training as demonstrated by a significant main effect of days, F(9,198)⫽ 26.354, P⬍0.001. Treatment with orexin-saporin impaired long-term memory as verified by a significant main effect of

265

treatment, F(2,22)⫽32.483, P⬍0.001, and an interaction of days and treatment that approached significance, F(18,198)⫽1.607, P⫽0.061. Post hoc analysis showed that both drug treatment groups differed significantly from the saline-treated rats, taking longer to reach the platform on the first trial of each day. Orexin-treated groups did not differ from one another. To determine the effect of orexin-saporin on short-term reference memory, the mean escape latency for each trial on day 1 was examined (Fig. 4). Only data from day 1 were used because results from subsequent days are influenced by training on previous days. Performance improved within the session as demonstrated by a significant effect of trials, F(5,110)⫽13.133, P⬍0.001. Treatment with orexin-saporin significantly impaired performance, F(2,22)⫽ 4.322, P⫽0.026. Post hoc analysis showed that orexin-100 and orexin-200 differed significantly from the saline control rats, but not from each other. Treatment and trials did not interact, F(10,110)⫽1.548, P⫽0.132, although the data suggest that such an interaction was present (Fig. 4). The lack of a treatment⫻trials interaction may be due to limited statistical power. Three different measures were used to assess the performance of rats on probe trials: time to platform location, number of platform crossings, and time in target quadrant (Fig. 5). Rats spent significantly greater time in the target quadrant on day 11 than on day 4: main effect of day, F(1,22)⫽15.372, P⫽0.001. Orexin-saporin tended to impair memory as the main effect of treatment approached significance, F(2,22)⫽3.032, P⫽0.069. Days did not interact with treatment, F(2,22)⫽0.147, P⫽0.864. 60

60 55 50 45 40 35 30 25 20 15 10 5 0

Saline Orexin 100 Orexin 200

Escape Latency (s)

Escape Latency (s)

50

40

30

20

Saline 10

Orexin 100 Orexin 200

0

1

2

3

4

5

6

7

8

9

10

Session Fig. 3. Mean escape latency for the first trial of each daily water maze session. In order to examine the effects of orexin-saporin on long-term memory, the first trial of each session was analyzed. Although no difference was observed on the first trial of the first session, rats treated with orexin-saporin took significantly longer than the salinetreated group to reach the hidden platform on the first trial of subsequent sessions. No difference was observed between rats treated with the two concentrations of orexin-saporin.

1

2

3

4

5

6

Trial Fig. 4. Mean escape latency for trials 1– 6 on the first day of water maze testing. In order to assess the effects of orexin-saporin on short-term reference memory, escape latencies for each trial of day 1 were compared. A significant main effect of treatment was observed, suggesting that orexin-saporin treatment impaired short-term memory. Although it appears that orexin-saporin treatment reduced the rate at which animals learned in the session, the interaction between treatment groups and trials was not observed.

266

H. R. Smith and K. C. H. Pang / Neuroscience 132 (2005) 261–271

Fig. 5. Performance on probe trials. The ability to remember the location of a hidden platform was tested on probe trials given at two different times in training (days 4 and 11). For the probe trial, the platform was removed from the water maze and three measures were used to assess memory for the platform location. Time to platform location was the time required to swim over the platform location; this measure is similar to escape latency except for the inability to escape due to the removal of the platform. Number of platform crossings is the number of times the rat swam over the platform location during the probe trial (60 s duration). Time in the target quadrant is the amount of time the rat spent in the quadrant where the platform was located. Quadrants were constructed so that the platform location was centered in the target quadrant. The time spent in any one quadrant expected by chance is 15 s. The main effect of intraseptal orexin-saporin was observed on the first day of probe trial (day 4) where differences between treatment groups were statistically different for time to platform location and approached significance for number of platform crossings. Time in target quadrant was not statistically different between groups. Treatment groups did not differ for any of the probe trial measures on day 11, demonstrating that sufficient training can reduce an impairment caused by orexin-saporin.

Rats crossed the platform location more often on day 11 than on day 4, suggesting that they had a better memory of the location of the platform later in training. The main effect of days between the two probe trials was significant, F(1,22)⫽26.841, P⬍0.001. A significant difference in performance between treatment groups was observed, F(2,22)⫽3.421, P⫽0.05; however, days and treatment did not interact, F(2,22)⫽0.75, P⫽0.484. This pattern of results suggests that all groups learned at similar rates between day 4 and day 11, despite an overall performance impairment of orexin-saporin-treated rats. Differences between the saline group and the orexin-200 group approached significance as revealed by post hoc analysis

(P⫽0.055). The orexin-100-treated animals were not different from either saline animals or orexin-200-treated animals. Finally, the time to first cross over the platform location (latency time) decreased significantly between probe trials, F(1,22)⫽41.492, P⬍0.001, suggesting that rats had a better memory of the location of the platform at the end of training as compared with early in training. Orexin-saporin impaired performance on this measure of the probe trials, as demonstrated by a main effect of treatment, F(2,22)⫽ 6.030, P⫽0.008, and an interaction between probe days and treatment groups, F(2,22)⫽4.934, P⫽0.017. Post hoc analysis indicated a significant difference in escape la-

H. R. Smith and K. C. H. Pang / Neuroscience 132 (2005) 261–271

267

tency time between the orexin-200 group and the saline group. The orexin-100-treated rats were not different from either saline- or orexin-200-treated rats. Orexin-saporin impaired performance in the first probe trial (day 4), suggesting a learning impairment existed early in training on the water maze task. This impairment was observed for latency time [F(2,24)⫽7.314, P⫽0.004] and platform crossings [F(2,24)⫽3.344, P⫽0.054], but not time in target quadrant [F(2,24)⫽2.162, P⫽0.139]. Post hoc analysis of the latency time measure showed that both orexin-100 and orexin-200 groups were significantly different from saline controls. For the probe on day 11, all groups performed similarly in all three measures, suggesting that all groups had learned the location of platform following 10 days of training. There was no main effect of treatment on latency times, F(2,24)⫽2.016, P⫽0.157, number of platform crossings, F(2,24)⫽2.196, P⫽0.135, or time spent in target quadrant, F(2,24)⫽0.868, P⫽0.434. In summary, acquisition of the water maze task was impaired with intraseptal administration of orexin-saporin at both concentrations. The orexin-treated groups took significantly longer to reach the platform on the first trial of each day, revealing a deficit in long-term reference memory. Additionally, orexin-treated rats were slower to acquire the location of the platform during the first session, demonstrating impaired short-term memory. Probe trials also demonstrated an impairment following orexin-saporin treatment with the deficits seen early, but not late, in training. Spontaneous alternation To determine the effect of orexin-saporin on short-term working memory, the mean percentage of alternations on a plus maze were computed for each group (Fig. 6A). Orexin-saporin decreased the percentage of alternations as demonstrated by a significant difference between treatment groups using a one-way ANOVA, F(2,23)⫽5.438, P⫽0.013. Post hoc analysis found a difference between the orexin-200 group and the other treatment groups, but no difference between the orexin-100 group and the saline controls. In contrast, intraseptal administration of orexinsaporin did not alter the exploration of the plus maze (Fig. 6B). Treatment groups did not differ in the number of arms entered, F(2,23)⫽.815. Therefore, rats treated with orexin200, but not orexin-100, were impaired on this short-term working memory task. However, neither dose of orexinsaporin altered exploration of the maze.

DISCUSSION In the present study, rats were administered different concentrations of orexin-saporin into the MSDB. Orexin-100 destroyed the majority of the GABAergic septohippocampal neurons as measured by a loss of PV-ir neurons, but preserved most of the cholinergic MSDB neurons as measured by sparing of ChAT-ir neurons. Orexin-200 almost completely destroyed the PV-ir neurons and many of the cholinergic neurons in the MSDB. These animals were

Fig. 6. Mean percentage of alternations (A) and total number of entries (B) on a spontaneous alternation task. Rats administered orexin-saporin into the MSDB alternated significantly less than salinetreated rats (A). Post hoc analysis revealed that rats treated with 200 ng/␮l were significantly impaired, while rats treated with 100 ng/␮l were similar to control rats. However, the number of entries into maze arms was not statistically different between treatment groups (B).

tested on two spatial memory tasks, the water maze and the spontaneous alternation task. Both groups were impaired with respect to saline control animals on the acquisition of the water maze task. This impairment was especially evident in the first trial of each daily session, reflecting impaired long-term memory. Additionally, treatment groups performed worse during the first session, suggesting an impairment of short-term memory. Performance on the

268

H. R. Smith and K. C. H. Pang / Neuroscience 132 (2005) 261–271

probe trials of the water maze was impaired following treatment with orexin-200, and the performance of orexin100 animals fell between saline control animals and orexin-200 animals. Orexin-200 also impaired spontaneous alternation, while no impairment was observed with orexin-100. The results suggest that orexin afferents to the medial septum may modulate spatial reference and working memory. Anatomy Damage following administration of 100 and 200 ng/␮l orexin-saporin was limited to the MSDB. At the highest concentration of orexin-saporin (300 ng/␮l), extensive tissue damage was observed of the medial septal area. The MSDB was shrunken and occasional holes were observed in the tissue. Associated with these effects was enlargement of the lateral ventricles. Because it was likely that orexin-saporin at this highest concentration caused damage to fibers of passage, behavioral data involving the orexin-300 treatment group were not reported. Intraseptal administration of orexin-200 destroyed most of the PV-ir neurons and many of the ChAT-ir neurons in the medial septal area, similar to a previous report (Gerashchenko et al., 2001). Both cholinergic and PV-ir neurons that were destroyed in this study are the populations of MSDB neurons known to project to the hippocampus (Kiss et al., 1990; Freund, 1998). Similar lesions have also been described following intraseptal ibotenic acid administration (Hepler et al., 1985a, b; Hagan et al., 1988). Intraseptal orexin-saporin at a concentration of 100 ng/␮l destroyed mainly PV-ir neurons without noticeable damage to cholinergic neurons, resulting in a relatively selective damage of GABAergic septohippocampal neurons. Therefore, our secondary aim to determine the mnemonic effects of partial damage to both cholinergic and GABAergic septohippocampal neurons could not be addressed. Orexin-100 produced a lesion in the MSDB that was similar to the damage produced by low doses of kainic acid (Malthe-Sorenssen et al., 1980; Pang et al., 2001). At present, we do not have an explanation for the greater damage of PV-ir neurons relative to ChAT-ir. Previous studies using higher concentrations have shown damage to both cholinergic and GABAergic septohippocampal neurons (Gerashchenko et al., 2001). However, the relative distribution of the hypocretin-2 receptor on MSDB neurons is not known, and may help to explain the differential sensitivity. It should be noted that we and others have only examined cholinergic and PV-ir MSDB neurons following intraseptal orexin-saporin. Whether non-PV, non-cholinergic neurons in the MSDB are damaged is an important, but still unknown, question that needs further investigation. Behavior All treatment groups demonstrated an ability to learn the location of the hidden platform across the 10-day training period; however, acquisition of the water maze task was impaired by both concentrations of orexin-saporin. The large impairment in performance on the first trial of each day suggests that the orexin-treated rats had an impaired

long-term memory, such as the memory required to remember the location of the hidden platform over 24 h. Orexin-treated groups also did not show the same type of improvement during the first session as saline-treated rats, suggesting an impairment of short-term memory. Therefore, the acquisition data suggest that the orexin-treated animals are impaired in both long-term and short-term reference memory. Results from the probe trials also suggest an impairment of memory. Separate analysis of day 4 and day 11 demonstrate an impairment early in training (day 4) for rats treated with orexin-saporin. All groups performed similarly on the last probe trial (day 11), which is consistent with previous studies involving selective and non-selective lesions of the MSDB (Pang et al., 2001). These results suggest that with sufficient training, rats with MSDB lesions can perform normally on spatial tasks. The impairment following orexin-200 treatment is consistent with previously reported memory deficits following lesions of both cholinergic and non-cholinergic neurons in the MSDB (Hagan et al., 1988; Pang et al., 2001). However, the impairment following orexin-100, which produced a relatively selective damage of GABAergic septohippocampal neurons, contrasts with our previous study using intraseptal kainic acid to produce selective lesions of the GABAergic septohippocampal neurons (Pang et al., 2001). Kainic acid lesions did not impair performance on a water maze or radial maze (Pang et al., 2001). Thus, the similar lesions produced by the two drugs, kainic acid and orexinsaporin (100 ng/␮l), result in two different behavioral results. Several possibilities may account for this discrepancy. First, the actual damage to cholinergic neurons may differ between kainic acid and orexin-saporin. Based on estimates of ChAT-ir neurons in the MSDB, orexin-saporin and kainic acid appeared to produce qualitatively similar types of damage. However, it is possible orexin-saporin impairs the ability of cholinergic neurons to function properly, whereas kainic acid does not alter cholinergic function. If this were the case, the impaired function of cholinergic neurons together with loss of GABAergic septohippocampal neurons may be sufficient to impair memory following orexin-saporin, similar to other treatments that damage both septohippocampal populations (Pang et al., 2001). Second, it is possible that kainic acid does not actually destroy the GABAergic septohippocampal neurons. Perhaps, kainic acid only stops the production of PV but does not destroy GABAergic septohippocampal neurons, explaining why no behavioral impairment is observed. We think that this scenario is unlikely considering that kainic acid was also shown to reduce some GAD-ir neurons in the MSDB (Pang et al., 2001) and decrease GAD activity (Malthe-Sorenssen et al., 1980). Third, the two chemicals used in the studies may have different effects on neuronal populations other than PV-ir and ChAT-ir neurons in the MSDB. Orexin-saporin, but not kainic acid, might destroy MSDB interneurons neurons. Because interneurons terminate on ChAT-ir neurons, orexin-saporin may impair the function of both PV-ir and ChAT-ir neurons, resulting in memory impairments. Alternatively, orexin-saporin might

H. R. Smith and K. C. H. Pang / Neuroscience 132 (2005) 261–271

damage peptidergic neurons, while kainic acid preserves these neurons. This neuronal population could play a role in spatial memory. Future studies using markers for non-PV-ir, non-ChAT-ir MSDB neurons are necessary to provide evidence that orexin-saporin and kainic acid target and destroy similar or different populations of neurons. Finally, damage to brain regions other than the MSDB could occur with intraseptal treatment using orexin-saporin, resulting in impairments in learning and memory. Afferents to the MSDB with hypocretin-2 receptors may take up the orexinsaporin and retrograde transport of the toxin might result in damage to these neurons. One possibility is the lateral hypothalamic orexin neurons that project to MSDB. However, direct injection of orexin-saporin into the lateral hypothalamus resulted in changes in sleep rhythms, but injection into the medial septal region did not produce the same effect, suggesting that injections into the medial septum preserved the activity of orexin neurons in the lateral hypothalamus (Gerashchenko et al., 2001, 2003). A differential impairment between orexin-100 and orexin-200 groups was observed on the spontaneous alternation task. The orexin-200 group had significantly less alternations than the orexin-100 group, even though the total number of arm entries was similar for all groups. The behavioral results together with the anatomical findings support the idea that cholinergic MSDB neurons, but not PV-ir neurons, are necessary for spatial working memory. This proposal has support from previous studies. Rats with intraseptal kainic acid lesions, which damage PV-ir but not cholinergic MSDB neurons, are not impaired on the eightarm radial maze (Pang et al., 2001). In addition, Chang and Gold (2004) showed an impairment on the spontaneous alternation task after treatment with 192-IgG-saporin. Finally, McIntyre et al. (2002) reported increases in hippocampal acetylcholine while performing the spontaneous alternation task. These results support the case that cholinergic MSDB neurons are necessary for spatial working memory. However, this conclusion argues against the idea presented above that orexin-100 might impair water maze performance by interfering with cholinergic, as well as GABAergic, function. Other studies have found that damage to cholinergic MSDB neurons using 192-IgG saporin does not impair spatial working memory (Baxter et al., 1995; McMahan et al., 1997; Chappell et al., 1998; Pang and Nocera, 1999). The difference between spontaneous exploration in the spontaneous alternation task and reinforced behavior in the radial maze may account for some of the differences in the results. Another possibility is that damage to both cholinergic and PV-ir neurons may be necessary to observe the memory impairments, but that damage to only the cholinergic neurons is insufficient to produce the deficits (Pang et al., 2001; Parent and Baxter, 2004). The lack of a spatial working memory impairment in the orexin-100 group may be due to more sparing of GABAergic neurons in the diagonal band as compared with the orexin200 group. The spared neurons project predominantly to the temporal pole of the hippocampus (Gaykema et al., 1990). However, previous studies have suggested that the

269

temporal pole of the hippocampus is not important in spatial memory, although most of the behavioral evidence for this view comes from water maze studies and may be specific to spatial reference memory (Moser and Moser, 1998). Less is known about the importance of temporal hippocampal areas in spatial working memory so this possibility still needs to be explored. The results from the spontaneous alternation task appear to differ from some of the findings using the water maze task. In particular, orexin-100-treated animals demonstrated impairments in both short-term and long-term memory components of the water maze task, but were not impaired on the spontaneous alternation task. However, accurate performance on the two tasks requires different types of memory. The water maze task requires reference memory where information regarding the platform location can be used over trials and days. In contrast, the spontaneous alternation task requires the animal to make a list of arms that were visited during individual sessions. Thus, it is not surprising that different results were observed in the two tasks for orexin-100. Differences in performance may also be due to variations in task difficulty. Spontaneous alternation in the four-arm plus maze may be inherently easier than the water maze so that rats treated with orexin100 were not sufficiently impaired to exhibit performance decrements on the spontaneous alternation task. If this were the case, increasing the task difficulty of the spatial working memory task, as in an eight-arm radial maze, might elicit impairments following orexin-100. In summary, our results suggest that spatial reference memory, but not working memory, is impaired following intraseptal orexin-100. Intraseptal treatment with 200 ng/␮l orexin-saporin eliminates hippocampal ␪ rhythm (Gerashchenko et al., 2001). The memory impairment associated with orexin200 in the present study supports the idea that ␪ rhythm is important for memory (Winson, 1978; Givens and Olton, 1990; Kahana et al., 1999; Hasselmo et al., 2002). However, our results showing an impairment of spatial reference memory with 100 ng/␮l orexin-saporin suggest that this dose should also completely eliminate hippocampal ␪ rhythm. Future studies will be needed to address this question. One potential problem with the present study is that damage to MSDB cells with orexin-2 (hypocretin-2) receptors might disrupt functions that are unrelated to those associated with the orexin system. This scenario might occur if cells with orexin-2 receptors are involved in both mnemonic and non-mnemonic processes with orexin afferents modulating a non-mnemonic process. Damage to the orexin-2 receptor containing cells would result in memory impairments, even though the orexin system might not have a direct role in memory. Despite this limitation, the present study represents the first attempt at investigating the function of orexin afferents to the MSDB. Future studies aimed at interfering with the actions of endogenous orexin in the MSDB are the next step to identifying an important role of the orexin system in memory. In summary, the present study demonstrates that orexin-saporin in different concentrations produces different

270

H. R. Smith and K. C. H. Pang / Neuroscience 132 (2005) 261–271

types of damage to the MSDB. A concentration of 100 ng/␮l damaged GABAergic septohippocampal neurons with relative sparing of cholinergic septal neurons. In contrast, a concentration of 200 ng/␮l resulted in a nonselective lesion of both cholinergic and GABAergic septohippocampal neurons in the MSDB. Impairments in acquisition of a water maze task were evident in rats treated with both concentrations of orexin-saporin. This impairment was apparent in trials requiring long-term, as well as short-term, reference memory. However, in a spontaneous alternation task, deficits were only seen in the rats with nonselective lesions of both cholinergic and GABAergic septohippocampal neurons. Our findings suggest that orexin afferents to the MSDB may have an important role in spatial memory. Together, results from present and previous studies suggest that orexin neurons may modulate memory by exciting cholinergic and GABAergic septohippocampal neurons (Wu et al., 2002, 2004). Acknowledgments—The authors would like to thank Ryan Yoder, J. P. Miller, Leslie Gulvas and Kelly Wright for their comments on an earlier version of the manuscript, and Bridget Bell for her assistance in the animal surgeries. This work was supported by funding from the National Institutes of Health (AG20560 and NS44373) and the generous contributions of Mrs. Dorothy Price.

REFERENCES Bannon AW, Curzon P, Gunther KL, Decker MW (1996) Effects of intraseptal injection of 192-IgG-saporin in mature and aged Long Evans rats. Brain Res 718:25–36. Baxter MG, Bucci DJ, Wiley RG, Gorman LK, Gallagher M (1995) Selective immunotoxic lesions of basal forebrain cholinergic cells: effects on learning and memory in rats. Behav Neurosci 109: 714 –722. Becker JT, Walker JA, Olton DS (1980) Neuroanatomical bases of spatial memory. Brain Res 200:307–320. Berger-Sweeney J, Heckers S, Mesulam MM, Wiley RG, Lappi DA, Sharma M (1994) Differential effects on spatial navigation of immunotoxin-induced cholinergic lesions of medial septal area and nucleus basalis magnocellularis. J Neurosci 14:4507– 4519. Cahill JFX, Baxter MG (2001) Cholinergic and noncholinergic septal neurons modulate strategy selection in spatial learning. Eur J Neurosci 14:1856 –1864. Cassel JC, Cassel S, Galani R, Kelche C, Will B, Jarrad L (1998) Fimbria-fornix vs. selective hippocampal lesions in rats: effects on locomotor activity and spatial learning and memory. Neurobiol Learn Mem 69:22– 45. Chang Q, Gold PE (2004) Impaired and spared cholinergic functions in the hippocampus after lesions of the medial septum/vertical limb of the diagonal band with 192 IgG-saporin. Hippocampus 14:170 –179. Chappell J, McMahan R, Chiba A, Gallagher M (1998) A re-examination of the role of basal forebrain cholinergic neurons in spatial working memory. Neuropharmacology 37:481–487. Dornan WA, McCampbell AR, Tinkler GP, Hickman LJ, Bannon AW, Decker MW, Gunther KL (1996) Comparison of site-specific injections into the basal forebrain on water maze and radial arm maze performance in the male rat after immunolesioning with 192 IgG saporin. Behav Brain Res 82:93–101. Dunnett SB, Everitt BJ, Robbins TW (1991) The basal forebraincortical cholinergic system: interpreting the functional consequences of excitotoxic lesions. Trends Neurosci 14:494 –500. Everitt BJ, Robbins TW (1997) Central cholinergic systems and cognition. Annu Rev Psychol 48:649 – 684.

Freund TF (1989) GABAergic septohippocampal neurons contain parvalbumin. Brain Res 478:375–381. Freund TF, Antal M (1988) GABA-containing neurons in the septum control inhibitory interneurons in the hippocampus. Nature 336: 170 –173. Gaykema RP, Luiten PGM, Nyakas C, Traber J (1990) Cortical projection patterns of the medial septum-diagonal band complex. J Comp Neurol 293:103–124. Geisser S, Greenhouse SW (1958) An extension of Box’s results on the use of the F distribution in multivariate analysis. Ann Math Stat 29:885– 891. Gerashchenko D, Blanco-Centurion C, Greco MA, Shiromani PJ (2003) Effects of lateral hypothalamic lesion with the neurotoxin hypocretin-2-saporin on sleep in Long-Evans rats. Neuroscience 116:223–235. Gerashchenko D, Salin-Pascual R, Shiromani P (2001) Effects of hypocretin-saporin injections into the medial septum on sleep and hippocampal theta. Brain Res 913:106 –155. Givens BS, Olton DS (1990) Cholinergic and GABAergic modulation of medial septal area: effect on working memory. Behav Neurosci 104:849 – 855. Hagan JJ, Salamone JD, Simpson J, Iversen SD, Morris RGM (1988) Place navigation in rats is impaired by lesions of the medial septum and diagonal band but not nucleus basalis magnocellularis. Behav Brain Res 27:9 –20. Hasselmo ME, Bodelon C, Wyble BP (2002) A proposed function for hippocampal theta rhythm: separate phases of encoding and retrieval enhance reversal of prior learning. Neural Comp 14:793–817. Heckers S, Ohtake T, Wiley RG, Lappi DA, Geula C, Mesulam M (1994) Complete and selective cholinergic denervation of rat neocortex and hippocampus but not amygdala by an immunotoxin against the p75 NGF receptor. J Neurosci 14:1271–1289. Hepler DJ, Olton DS, Wenk GL, Coyle JT (1985a) Lesions in nucleus basalis magnocellularis and medial septal area of rats produce qualitatively similar memory impairments. J Neurosci 5(4): 866 – 873. Hepler DJ, Wenk GL, Cribbs BL, Olton DS, Coyle JT (1985b) Memory impairments following basal forebrain lesions. Brain Res 346: 8 –14. Jakab RL, Leranth C (1995) Septum. In: The rat nervous system (Paxinos G, ed), pp 405– 422. San Diego: Academic Press. Janis LS, Glasier MM, Fulop Z, Stein DG (1998) Intraseptal injections of 192 IgG saporin produce deficits for strategy selection in spatialmemory tasks. Behav Brain Res 90:23–34. Johnson DA, Zambon NJ, Gibbs RB (2002) Selective lesion of cholinergic neurons in the medial septum by 192 IgG-saporin impairs learning in a delayed matching to position T-maze paradigm. Brain Res 943:132–141. Kahana MJ, Sekuler R, Caplan JB, Kirschen M, Madsen JR (1999) Human theta oscillations exhibit task dependence during virtual maze navigation. Nature 399:781–784. Kesner RP, Crutcher KA, Measom MO (1986) Medial septal and nucleus basalis magnocellularis lesions produce order memory deficits in rats which mimic symptomatology of Alzheimer’s disease. Neurobiol Aging 7:287–295. Kilduff TS, Peyron C (2000) The hypocretin/orexin ligand-receptor system: implications for sleep and sleep disorders. Trends Neurosci 23:359 –365. Kiss J, Patel AJ, Freund TF (1990) Distribution of septohippocampal neurons containing parvalbumin or choline acetyltransferase in the rat brain. J Comp Neurol 298:362–372. Lamprea MR, Cardenas FP, Silveira R, Morato S, Walsh TJ (2000) Dissociation of memory and anxiety in a repeated elevated plus maze paradigm: forebrain cholinergic mechanisms. Behav Brain Res 117:97–105. Leanza G, Nilsson OG, Wiley RG, Bjorklund A (1995) Selective lesioning of the basal forebrain cholinergic system by intraventricular

H. R. Smith and K. C. H. Pang / Neuroscience 132 (2005) 261–271 192 IgG-saporin: behavioral, biochemical and stereological studies in the rat. Eur J Neurosci 7:329 –343. Lehmann O, Jeltsch H, Lazarus C, Tritschler L, Bertrand F, Cassel JC (2002) Combined 192 IgG-saporin and 5,7-dihydroxytryptamine lesions in the male rat brain: a neurochemical and behavioral study. Pharmacol Biochem Behav 72:899 –912. Malthe-Sorenssen D, Odden E, Walaas I (1980) Selective destruction by kainic acid of neurons innervated by putative glutamergic afferents in septum and nucleus of diagonal band. Brain Res 182: 461– 465. Markowska AL, Wenk GL, Olton DS (1990) Nucleus basalis magnocellularis and memory: differential effects of two neurotoxins. Behav Neural Biol 54:13–26. McDonald RJ, White NM (1994) Parallel information processing in the water maze: evidence for independent memory systems involving dorsal striatum and hippocampus. Behav Neur Biol 61:260 –270. McIntyre K, Shanthi NP, Marriott LK, Gold PE (2002) Competition between memory systems: acetylcholine release in the hippocampus correlates negatively with good performance on an amygdaladependent task. J Neurosci 22:1171–1176. McKinney M, Coyle JT, Hedreen JC (1983) Topographic analysis of the innervation of the rat neocortex and hippocampus by the basal forebrain cholinergic system. J Comp Neurol 217:103–121. McMahan RW, Sobel TJ, Baxter MG (1997) Selective immunolesions of hippocampal cholinergic input fail to impair spatial working memory. Hippocampus 7:130 –136. Mizumori SJY, Perez GM, Alvarado MC, Barnes CA, McNaughton BL (1990) Reversible inactivation of the medial septum differentially affects two forms of learning in rats. Brain Res 528:12–20. Morris RGM, Garrud P, Rawlins JNP, O’Keefe J (1982) Place navigation impaired in rats with hippocampal lesions. Nature 297: 681– 683. Moser MB, Moser EI (1998) Functional differentiation in the hippocampus. Hippocampus 8:608 – 619. Nambu T, Sakurai T, Mizukami K, Hosoya Y, Yanagisawa M, Goto K (1999) Distribution of orexin neurons in the adult rat brain. Brain Res 827:243–260. Olton DS, Becker JT, Handelmann JE (1979) Hippocampus, space and memory. Behav Brain Sci 2:313–365. Packard MG, McGaugh JL (1996) Inactivation of hippocampus or caudate nucleus with lidocaine differentially affects expression of place and response learning. Neurobiol Learn Mem 65:65–72. Pang KCH, Nocera R (1999) Interactions between 192-IgG-saporin and intraseptal cholinergic and GABAergic drugs: role of cholin-

271

ergic medial septal neurons in spatial working memory. Behav Neurosci 113:265–275. Pang KCH, Nocera R, Secor AJ, Yoder RM (2001) GABAergic septohippocampal neurons are not necessary for spatial memory. Hippocampus 11:814 – 827. Parent MB, Baxter MG (2004) Septohippocampal acetylcholine: involved in but not necessary for learning and memory? Learn Mem 11:9 –20. Robbins TW, Everitt BJ, Marston HM, Wilkerson J, Jones GH, Page KJ (1989) Comparative effects of ibotenic acid- and quisqualic acidinduced lesions of the substantia innominata on attentional function in the rat: further implications for the role of the cholinergic neurons of the nucleus basalis in cognitive processes. Behav Brain Res 35:221–240. Wainer BH, Levey AI, Rye DB, Mesulam MM, Mufson EJ (1985) Cholinergic and non-cholinergic septohippocampal pathways. Neurosci Lett 54:45–52. Waite JJ, Thal LJ (1996) Lesions of the cholinergic nuclei in the rat basal forebrain: excitotoxins vs. an immunotoxin. Life Sci 58: 1947–1953. Walaas I (1981) The effects of kainic acid injections on guanylate cyclase activity in the rat caudatoputamen, nucleus accumbens, and septum. J Neurosci 36:233–241. Walsh TJ, Herzog CD, Gandhi C, Stackman RW, Wiley RG (1996) Injection of IgG 192-saporin into the medial septum produces cholinergic hypofunction and dose-dependent working memory deficits. Brain Res 726:69 –79. Wiley RG, Oeltmann TN, Lappi DA (1991) Immunolesioning: selective destruction of neurons using immunotoxin to rat NGF receptor. Brain Res 562:149 –153. Winson J (1978) Loss of hippocampal theta rhythm results in spatial memory deficits in the rat. Science 201:160 –163. Wrenn CC, Lappi DA, Wiley RG (1999) Threshold relationship between lesion extent of the cholinergic basal forebrain in the rat and working memory impairment in the radial maze. Brain Res 847: 284 –298. Wu M, Zongming Z, Leranth C, Xu C, van de Pol AN, Alreja M (2002) Hypocretin increases impulse flow in the septohippocampal GABAergic pathway: implications for arousal via a mechanism of hippocampal disinhibition. J Neurosci 22:7754 –7765. Wu M, Zaborszky L, Hajszan T, van de Pol AN, Alreja M (2004) Hypocretin/orexin innervation and excitation of identified septohippocampal cholinergic neurons. J Neurosci 24:3527–3536.

(Accepted 31 December 2004) (Available online 12 March 2005)