The Journal of Neuroscience, October 1994, 14(10): 59885995 Behavioral, Biochemical, Histological, and Electrophysiological Effects of 192 IgG-Saporin Injections into the Basal Forebrain of Rats Gary L. Wenk,’ James D. Stoehr,’ George Quintana,’ Sherri Mobley,’ and Ronald G. Wiley2 ‘Arizona Research Laboratories, Division of Neural Systems, Memory and Aging, University of Arizona, Tucson, Arizona 85724 and 2DVAMC and Vanderbilt University, Nashville, Tennessee 37232 The behavioral, biochemical, histological, and electrophys- layed-alternation or passive avoidance tasks were not im- iological effects of a basal forebrain injection of saporin, a paired by the lesions. ribosome-inactivating protein, coupled to a monoclonal an- These data confirm the effectiveness and specificity of tibody against the low-affinity NGF receptor (192 IgG) were this novel lesioning tool and suggest that selective loss of investigated in adult rats. Within the basal forebrain region, NBM cholinergic cells is not sufficient to impair performance the low-affinity NGF receptor is exclusively expressed by in these behavioral tasks. cholinergic neurons in the medial septal area, diagonal band, [Key words: 192 IgG-saporin, nucleus basalis, EEG, basal and nucleus basalis magnocellularis (NBM). The presence forebrain, lesion, T-maze alternation, rats] of this receptor upon these cells confers a degree of spec- ificity to the 192 IgG-saporin that could not previously be Loss of neurons in the basal forebrain may play an important achieved by previous lesioning techniques, such as excit- role in the cognitive deficits associatedwith aging and Alzhei- atory amino acids. mer’s disease(Bartus et al., 1982). The basal forebrain region Rats with unilateral injections of different amounts of 192 includes the medial septum, vertical and horizontal limbs of the IgG-saporin were prepared to determine the optimal con- diagonalband of Broca, and the nucleusbasalis magnocellularis ditions in order to produce a lesion restricted to the NBM (NBM) (Mesulam et al., 1983). Basal forebrain lesionsproduce that would not destroy cholinergic afferents to hippocampus behavioral deficits in both monkeys and rats (for a review, see or nearby regions. Electroencephalographic (EEG) record- Olton and Wenk, 1987) and increasethe incidence of slow-wave ings were taken from these lesioned rats before and during activity in neocortical EEG recordings in rats (Stewart et al., treatment with scopolamine (1 mg/kg, i.p.). 1984; Buzsaki et al., 1988; Riekkinen et al., 1990a,b). These Another group of rats received bilateral NBM injections of lesionsare frequently produced by microinjection of glutamate 192 IgG-saporin and were behaviorally tested using a re- analogssuch as ibotenic (IBO) or quisqualic (QUIS) acid. Both warded, delayed-alternation task on a T-maze and a passive IBO and QUIS effectively destroy cholinergic cells within the avoidance task. Finally, histological and biochemical inves- basalforebrain, but only IBO impairs performancein behavioral tigations confirmed the effectiveness and specificity of the tasks that require learning and memory (Dunnett et al., 1987; 192 IgG-saporin. Etherington et al., 1987; Everitt et al., 1987; Markowska et al., The results showed that the 192 IgG-saporin did not de- 1990; Riekkinen et al., 1991; Wenk et al., 1992). This suggests stroy neurotensin, galanin, somatostatin, NADPH-diaphor- that the memory impairments are not related to the cholinergic ase, or neuropeptide Y neurons within the NBM. Also, biom- cell loss but may be related to the loss of noncholinergic cells arkers of cholinergic function were significantly decreased within the basal forebrain (Riekkinen et al., 1990a,b; Dunnett throughout the neocortex and within the NBM, but not in the et al., 1991; Sarter and Dudchenko, 1991; Wenk et al., 1992) olfactory bulbs, hippocampus, or dorsal caudate nucleus. such as GABA, substanceP (SP), neurotensin (NT), leu-en- lntraperitoneal injections of scopolamine, but not NBM in- kephalin (LE), somatostatin (SOM), galanin, or neuropeptideY jections of 192 IgG-saporin, increased total power across (NPY) (Candy et al., 1985). The resolution of the role of basal all frequency bands; however, slow-wave frequencies forebrain cholinergic cellsin mnemonic processeshas been ham- showed a greater increase in power as compared to fast- pered by the lack of a specific method for selectively destroying wave frequencies. Acquisition, and performance of the de- cholinergic neurons. The lack of specificity of IBO and QUIS has precluded a clear interpretation of the behavioral data. Received Nov. 29, 1993; revised Feb. 28, 1994; accepted Mar. 29, 1994. The nonspecificity of neurotoxins has also prevented a clear This work is dedicated to the memory of Dr. David S. Olton, whose guidance interpretation of the effects of basal forebrain lesionsupon neo- and inspiration made these studies possible. G.L.W. thanks Drs. Carol Barnes cortical EEG activity. Recent reports found a pronounced in- and Cindy Erickson for their valuable assistance with the EEG studies. and Ms. creasein large-amplitude, slow-wave activity predominating in Jennifer durney and Mr. Chad Livdahl for their excellent assistance in conducting the behavioral testing. This work was supported by The National Science Foun- the neocortex ipsilateral to a unilateral lesion (Stewart et al., dation, BNS 89-14941. 1984; Buzsaki et al., 1988; Riekkinen et al., 1990a,b; Vander- Correspondence should be addressed to Dr. Gary L. Wenk, Division of Neural Systems, Memory and Aging, 384 Life Sciences North, University of Arizona, wolf et al., 1993). While correlations were found between the Tucson, AZ 85724. increasein slow-waveactivity and cortical ChAT loss,the amount Copyright 0 1994 Society for Neuroscience 0270-6474/94/145986-10$05.00/O of incidental and nonspecific damage to noncholinergic fore- The Journal of Neuroscience, October 1994, f4(10) 5987 brain neurons may also underlie the changes in EEG. Therefore, struments, Tujunga, CA) with the incisor bar set 2.0 mm below the ear the use of a toxin that is specific for cholinergic neurons is bars (flat skull). The scalp was incised and retracted and holes were drilled in the skull with a dental drill. required in order to determine whether the loss of NBM cho- Unilateral lesion study. The results of these unilateral lesion studies linergic neurons is solely responsible for cortical EEG slowing. guided the choice of lesion coordinates for the behavioral studies that A powerful new approach to selective neural lesioning uses used bilateral lesions. The purpose of this study was to determine the antineuronal immunotoxins (Wiley, 1992). In the present study, optimal concentration of 192 IgG-saporin that would destroy the great- est number of NBM cholineraic cells with the least amount of cell loss basal forebrain cholinergic cells were selectively destroyed by in the horizontal limb of thediagonal band of Broca, the dorsal and injection of an antineuronal immunotoxin recently developed ventral aspects of the basal ganglia, and other surrounding structures by Wiley et al. (199 1). This compound consists of a monoclonal that may receive cholinergic innervation, for example, the olfactory antibody (192 IgG) to the low-affinity nerve growth factor (NGF’) bulbs. Our preliminary studies had already investigated the optimal receptor that has been armed with the ribosome-inactivating volume and anterior-posterior coordinates that would preclude exten- sive spread of the 192 IgG-saporin and injury to medial septal and protein saporin. Injections of this 192 IgG-saporin complex diagonal band neurons, respectively (data not shown). The unilateral produce long-lasting depletions in cholinergic markers through- lesion coordinates were as follows: 0.9 mm posterior to bregma, 2.8 out the forebrain (Book et al., 1992). The actual selectivity of mm lateral from the midline (left or right side), and 6.9 mm below the this molecule remains to be fully defined. In the only published dura for all rats. The amounts of 192 IgG-sanorin tested were 5, 10, and 22.5 ng injected in a constant total ;olume of 0.03 ~1. study, a single high-dose intracerebroventricular injection (4.69 Behavioral study. Bilateral lesion coordinates for the behavioral study pg of 192 IgG-saporin in 7.0 ~1) reduced ChAT activity through- were 0.9 mm posterior to dregma, 2.8 mm lateral from the midline, out the neocortex, hippocampus, and septal area (Nilsson et al., and 6.9 mm below the dura. The 192 IgG-saporin (10 ng in 0.03 ~1) 1992). Endogenous levels of dopamine were unaffected by in- was injected slowly (taking approximately 2 min) and the needle was jections of 192 IgG-saporin, while endogenous levels of nor- left in mace for 5 min to limit diffusion dorsal to the iniection site. This concentration of 192 IgG-saporin was chosen because the preliminary epinephrine were elevated in the hippocampus and striatum. unilateral lesion study outlined above found that this concentration The lesion significantly impaired acquisition of the Morris water provided the greatest loss of cholinergic cells that project to cortex, maze task. These data do not address the issue of which cho- without loss of hippocampal afferents, and with the most limited loss linergic neurons within the basal forebrain were responsible for of striatal cholinergic cells. Control rats had the injection cannula low- ered to the same coordinates but no injection was made. the behavioral impairments, for example, the NBM, which in- The following